Further use of protein kinase N beta

ABSTRACT

The present invention is related to use of protein kinase N beta or a fragment or derivative thereof as a downstream target of the PI 3-kinase pathway, preferably as a downstream drug target of the PI 3-kinase pathway.

[0001] This application claims priority from U.S. provisionalapplication serial No. 60/409,570, filed Sep. 11, 2002, the entirety ofwhich is hereby incorporated by reference. The disclosure of EuropeanPatent Application No. 02018572.4, filed Aug. 14, 2002, for whichbenefit under 35 USC § 119 is claimed, also is expressly incorporatedherein in its entirety.

FIELD OF THE INVENTION

[0002] The invention provides compositions and methods related to theuse of protein kinase N beta.

BACKGROUND OF THE INVENTION

[0003] Modern drug development no longer relies on a more or lessheuristic approach but typically involves the elucidation of themolecular mechanisms underlying a disease or a condition, theidentification of candidate target molecules and the evaluation of saidtarget molecules. Once such a validated target molecule, which is hereinreferred to also as target, is available, drug candidates directedthereto may be tested. In many cases such drug candidates are members ofa compound library which may consist of synthetic or natural compounds.Also the use of combinatorial libraries is common. Such compoundlibraries are herein also referred to as candidate compound libraries.Although in the past this approach has proven to be successful, it isstill time and money consuming. A variety of technologies currently areapplied for target identification and target validation.

[0004] Still, numerous tumours and cancers pose a significant threat tohuman health. In order to create safer and more powerful drugs havingless side effects, it is necessary to identify target molecules which,upon being addressed by appropriate compounds, may specifically andselectively be influenced in their activity or presence. Because of thepreferably selective and specific interaction between the compound,which may be a potential or candidate drug, and the target, the target'sfunction in a disease or diseased condition such as, for example,cancer, tumorigenesis and metastasis, may be influenced and thus thedisease treated or prevented and the diseased condition ameliorated.

[0005] It is apparent that new targets suitable for development of newtherapeutic approaches in the treatment of tumorigenesis and cancer aregreatly to be desired. It also is apparent that new methods oftherapeutic intervention directed at those targets are greatly to bedesired.

SUMMARY OF THE INVENTION

[0006] It is therefore an object of the invention to provide new targetsfor therapeutic invention and the treatment of disease. It is a furtherobject of the invention to provide compositions and methods suitable fortherapeutic intervention against these new targets.

[0007] In accordance with these objects, there has been provided, inaccordance with a first aspect of the invention, the use of proteinkinase N beta or a fragment or derivative thereof as a downstream targetof the PI 3-kinase pathway, preferably as a downstream drug target ofthe PI 3-kinase pathway.

[0008] In accordance with a second aspect of the invention there areprovided compositions and methods for using protein kinase N beta or afragment or derivative thereof for the manufacture of a medicament forthe treatment and/or prevention of a disease and/or for the manufactureof a diagnostic agent for the diagnosis of a disease, whereby thedisease is selected from the group comprising cancers, metastaticcancers and any pathological conditions involving the PI 3-kinasepathway.

[0009] In an embodiment of the use according to the first and secondaspect of the present invention protein kinase N beta has an amino acidsequence according to SEQ ID NO. 1 or according to databank entry PIDg7019489 or databank entry gi 7019489, or a part or derivative thereof.

[0010] In accordance with a third aspect of the invention there isprovided a use of a nucleic acid coding for protein kinase N beta, or afragment or a derivative thereof for the treatment and/or prevention ofa disease and/or for the manufacture of a diagnostic agent for thediagnosis of a disease, whereby the disease is selected from the groupcomprising cancers, metastatic cancers and any pathological conditionsinvolving the PI 3-kinase pathway.

[0011] In an embodiment of the use according to the third aspect of thepresent invention protein kinase N beta has an amino acid sequenceaccording to SEQ ID NO. 1 or according to databank entry PID g7019489 ordatabank entry gi 7019489, or a part or derivative thereof.

[0012] In another embodiment of the use according to the third aspect ofthe present invention the nucleic acid is a nucleic acid according toSEQ ID NO. 2 or according to databank entries gi 7019488 or NM_(—)01335,preferably NM_(—)01335.1.

[0013] In another embodiment of the use according to the any of theaspects of the present invention protein kinase N beta is coded by anucleic acid according to SEQ ID NO. 2 or according to databank entriesgi 7019488 or NM_(—)01335, preferably NM_(—)01335.1.

[0014] In a preferred embodiment of the use according to the thirdaspect of the present invention the nucleic acid sequence, but for thedegeneracy of the genetic code, would hybridize to the inventive nucleicacid subject to the third aspect of the present invention.

[0015] In a further embodiment of the use according to the any of theaspects of the present invention the nucleic acid sequence is a sequencewhich hybridizes under stringent conditions to the nucleic acid sequenceor part thereof, according to SEQ ID NO. 2 or according to databankentries gi 7019488 or NM_(—)01335, preferably NM_(—)01335.1.

[0016] In a preferred embodiment of the use according to any of theaspects of the present invention the disease is characterized such thatthe cells being involved in said disease, lack PTEN activity, show anincreased aggressive behaviour, or are cells of a late stage tumor.

[0017] In another preferred embodiment of the use according to the thirdaspect of the present invention the disease is a late stage tumor.

[0018] In accordance with another aspect of the invention there isprovided a method for the screening of an agent for the treatment and/orprevention of a disease and/or for the manufacture of a diagnostic agentfor the diagnosis of a disease, whereby the disease is selected from thegroup comprising cancers, metastatic cancers and any pathologicalconditions involving the PI 3-kinase pathway comprising the steps:

[0019] a) providing a candidate compound,

[0020] b) providing an expression system for protein kinase N betaand/or a system detecting the activity of protein kinase N beta;

[0021] c) contacting of the candidate compound with the expressionsystem for protein kinase N beta and/or the system detecting activity ofprotein kinase N beta;

[0022] d) determining if the expression and/or the activity of proteinkinase N beta is changed under the influence of the candidate compound.

[0023] In an embodiment of the method according to the fourth aspect ofthe present invention the candidate compound is contained in a libraryof compounds.

[0024] In another embodiment of the method according to the fourthaspect of the present invention the candidate compound is selected fromthe group of classes of compounds comprising peptides, proteins,antibodies, anticalines, functional nucleic acids, natural compounds andsmall molecules.

[0025] In a preferred embodiment of the method according to the fourthaspect of the present invention the functional nucleic acids areselected from the group which comprises aptamers, aptazymes, ribozymes,spiegelmers, antisense oligonucleotides and siRNA.

[0026] In a further preferred embodiment of the method according to thefourth aspect of the present invention protein kinase N beta or thenucleic acid coding for protein kinase N beta are the ones described inconnection with any other aspect of the present invention.

[0027] In accordance with a fifth aspect of the invention there isprovided the use of protein kinase N beta or a part or derivativethereof and/or nucleic acid or a part or derivative thereof coding forprotein kinase N beta as target molecule for the development and/ormanufacture of a medicament for the treatment and/or prevention of adisease and/or for the manufacture of a diagnostic agent for thediagnosis of a disease, whereby the disease is selected from the groupcomprising cancers, metastatic cancers and any pathological conditionsinvolving the PI 3-kinase pathway.

[0028] In an embodiment of the use according to the fifth aspect of thepresent invention the medicament and/or the diagnostic agent comprisesan agent, which is selected from the group comprising antibodies,peptides, anticalines, small molecules, antisense molecules, aptameres,spiegelmers and RNAi molecules.

[0029] In a preferred embodiment of the use according to the fifthaspect of the present invention the agent interacts with the proteinkinase N beta or a part or derivative thereof.

[0030] In an alternative embodiment of the use according to the fifthaspect of the present invention the agent interacts with the nucleicacid coding for protein kinase N beta or a part or derivative thereof,in particular with MRNA, genomic nucleic acid or cDNA for protein kinaseN beta.

[0031] In accordance with a sixth aspect of the invention there isprovided the use of a polypeptide which interacts with protein kinase Nbeta or a part or derivative thereof, for the development or manufactureof a medicament for the treatment and/or prevention of a disease and/orfor the manufacture of a diagnostic agent for the diagnosis of adisease, whereby the disease is selected from the group comprisingcancers, metastatic cancers and any pathological conditions involvingthe PI 3-kinase pathway.

[0032] In an embodiment of the use according to the sixth aspect of thepresent invention the polypeptide is selected from the group whichcomprises antibodies against protein kinase N beta or a part orderivative thereof and polypeptides binding protein kinase N beta or apart or derivative thereof.

[0033] In accordance with a seventh aspect of the invention there isprovided the use of a nucleic acid which interacts with protein kinase Nbeta or a part or derivative thereof, for the development or manufactureof a medicament for the treatment and/or prevention of a disease and/orfor the manufacture of a diagnostic agent for the diagnosis of adisease, whereby the disease is selected from the group comprisingcancers, metastatic cancers and any pathological conditions involvingthe PI 3-kinase pathway.

[0034] In an embodiment of the use according to the seventh aspect ofthe present invention the nucleic acid is selected from the group whichcomprises aptamers and spiegelmers.

[0035] In accordance with an eighth aspect of the invention there isprovided the use of a nucleic acid which interacts with a nucleic acidcoding for protein kinase N beta or a part or derivative thereof, forthe development or manufacture of a medicament for the treatment and/orprevention of a disease and/or for the manufacture of a diagnostic agentfor the diagnosis of a disease, whereby the disease is selected from thegroup comprising cancers, metastatic cancers and any pathologicalconditions involving the PI 3-kinase pathway.

[0036] In an embodiment of the use according to the eighth aspect of thepresent invention the interacting nucleic acid is an antisenseoligonucleotide, a ribozyme and/or siRNA.

[0037] In a further embodiment of the use according to the eighth aspectof the present invention the nucleic acid coding for protein kinase Nbeta or a part or derivative thereof is the cDNA, mRNA or hnRNA.

[0038] In an embodiment of the use according to the eighth aspect of thepresent invention the protein kinase N beta and/or the nucleic acidcoding for protein kinase N beta is the one described in connection withany aspect of the present invention.

[0039] In accordance with a ninth aspect of the invention there isprovided a pharmaceutical composition comprising at least one agentselected from the group comprising protein kinase N beta or a part orderivative thereof, small molecules interacting with protein kinase Nbeta or a part or derivative thereof or with a nucleic acid coding forprotein kinase N beta or a part or derivative thereof, antibodiesspecific for protein kinase N beta or a part or derivative thereof,polypeptides interacting with protein kinase N beta or a part orderivative thereof, a nucleic acid coding for protein kinase N beta or apart or derivative thereof, nucleic acids interacting with proteinkinase N beta or a part or derivative thereof or nucleic acidsinteracting with a nucleic acid coding for protein kinase N beta or apart or derivative thereof, and at least one pharmaceutically acceptablecarrier, preferably for the prevention and/or the treatment of a diseasewhereby the disease is selected from the group comprising cancers,metastatic cancers and any pathological conditions involving the PI-3kinase pathway.

[0040] In accordance with a stenth aspect of the invention there isprovided a kit for the characterisation of a disease or a conditionwhich is selected from the group comprising cancers, metastatic cancersand any pathological conditions involving the PI-3 kinase pathway,comprising at least one agent which is selected from the groupcomprising protein kinase N beta or a part or derivative thereof,antibodies specific for protein kinase N beta or a part or derivativethereof, polypeptides interacting with protein kinase N beta or a partor derivative thereof, polypeptides interacting with a nucleic acidcoding for protein kinase N beta or a part or derivative thereof,nucleic acids interacting with protein kinase N beta or a part orderivative thereof, nucleic acids interacting with a nucleic acid codingfor protein kinase N beta or a part or derivative thereof, andoptionally at least one other compound.

BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The present invention is now further illustrated by the followingfigures and examples which are not intended to limit the scope ofprotection. From these figures and examples further features,embodiments and advantages may be taken, wherein

[0042]FIG. 1 shows a schematic representation of growth factor inducedactivation of the PI 3-kinase pathway. Growth factor stimulation ofcells leads to activation of their cognate receptors at the cellmembrane, which in turn associate with and activate intracellularsignalling molecules such as PI 3-kinase. The tumor suppressor PTENinterferes with PI 3-kinase mediated downstream responses and ensuresthat activation of the pathway occurs in a transient manner. LY294002 isa small molecule inhibitor of PI 3-kinase. One of the known downstreamgenes of PI 3-K is mTOR (mammalian target of Rapamycin) which can beinhibited by the clinically approved drug rapamycin (Rapamune). PI 3-Kis involved in the regulation of cell proliferation, cell survival,glucose transport, translation, metastasis and migration. X areindicating downstream effectors which represent potential drug targetsthat are predicted to be involved in promoting metastatic behavior ofcancer cells. This class of effector molecules which act furtherdownstream in the pathway are likely to represent better drug targetsthan more “upstream” targets such as mTOR, since they have fewerpleiotropic effects.

[0043]FIG. 2 shows the measurement of lymph node metastasis in anorthotopic PC-3 mouse model after treatment with rapamycin;

[0044]FIG. 3 shows the experimental approach to identify PKNbeta as adownstream drug target of the PI 3-Kinase pathway;

[0045]FIG. 4 shows a primary GeneBloc screen on PKNbeta;

[0046]FIG. 5 shows the growth of PC3 cells transfected with PKNbetaspecific GB on matrigel;

[0047]FIG. 6 shows RNA interference by transient expression of siRNA inHeLaB cells: (A) siRNA molecules were generated by promoter (U6+2)driven expression of target specific sequences (template derived fromgene of interest containing a 21-mer sense and reverse complementarysequences linked by 12-mer poly A stretch. Upon transcription RNAs arelikely to form double-stranded siRNA molecules; (B) Template sequencesof targeted genes for siRNA expression. Corresponding sequence wereintroduced into expression vectors carrying the U6+2 promoter cassette;(C) Effect of siRNA expression on cell growth and proliferation.Constructs (see above) were transiently expressed by transfection inHeLaB cells for RNAi interference experiments. Cells were harvested 48hour after transfected and subsequently seeded (80000 cells per well) on“matrigel” gel. The effect of RNA interference on the expression ofcorresponding genes was analyzed by assaying transfected cells forgrowth/proliferation on matrigel. Expression of siRNA targeted to PTENhad no affect on HeLaB cell growth on matrigel (right panel), whereasexpression of siRNA specific to p110beta and PKNbeta severely disturbedthe behaviour of HeLaB growth on matrigel (middle and right panels).;

[0048]FIG. 7 shows photographs of human prostate cells and humanprostate cancer cells upon hybridisation using protein kinase N betaantisense and sense sequences as probes;

[0049]FIG. 8 shows a diagram depicting the volume of primary tumors inan orthotopic prostate tumor model using two different siRNA constructs(FIG. 8A), a diagram depicting the volume-of lymph node metastases in anorthotopic prostate tumor model using two different siRNA constructs(FIG. 8B), and photographs of prostate and lymph nodes in an orthotopicprostate tumor model using control siRNA (FIG. 8C1) and a protein kinaseN beta specific siRNA construct (FIG. 8C2);

[0050]FIG. 9 shows a Western-blot analysis of different protein kinase Nbeta derivatives and their activities using MPB as a standardphosphorylation substrate upon transient overexpression in HeLa cellsand anti-protein kinase N beta antibody (anti-PK) for detecting therelative expression levels of the kinase derivatives (FIG. 9A), afurther Western-blot analysis of different protein kinase N betaderivatives using an antibody specific for the phosphorylated form ofprotein kinase N beta (FIG. 9B), and a schematic representation of thevarious protein kinase N beta derivatives used (FIG. 9C);

[0051]FIG. 10 shows a Western Blot of various protein kinase N betaderivatives (FIG. 10A) to monitor the expression levels thereof in HeLacells and a gel analysis of the phosphorylation of the protein kinase Nbeta derivatives (FIG. 10B);

[0052]FIG. 11 shows the results of immunoprecipitation assays to detectphosphorylation of protein substrates for protein kinase N betadetecting the phosphorylated form thereof by Western blotting (FIG. 11A)or the incorporation of ³²P-labelled phosphate by autoradiography (FIG.11B). To ensure that comparable amounts of PKNbeta were present in therespective immune precipitates the filter shown in FIG. 11A was reprobedusing an anti-PKNbeta antibody (“kinase”, FIG. 11C).

[0053]FIG. 12 shows a Western Blot analysis comparing the expression ofendogenous protein kinase N beta in samples that were treated withLY294002 for different times in HeLa and PC-3 cells. The level ofphosphorylated AKT was monitored in parallel to confirm efficacy of thePI 3-kinase inhibitor.

[0054]FIG. 13 shows the relative protein amounts and kinase activitiesof various recombinant PKNbeta derivatives present in immuno-complexes.The cells expressing the respective recombinant proteins had beentreated with he PI 3-kinase inhibitor LY294002 prior to lysis for theindicated times.

[0055]FIG. 14 shows a panel of pictures, whereby the cellulardistribution of PKN beta and derivatives thereof such as PKN betawildtype (FIG. 14A), PKN beta derivative TA (FIG. 14B), PKN betaderivative KE (FIG. 14C) and PKN beta deltaN (FIG. 14D) was investigatedby confocal fluorescence microscopy. HA-tagged recombinant derivativesof PKNbeta were transiently expressed in HeLa cells for 48 h. Afterfixing and permeabilization, expression of the recombinant proteins wasdetected by using an anti-HA antibody followed by an FITC-conjugatedanti-mouse antibody. The cells were counterstained by labelling thecytoskeletal actin with rhodamin-phalloidin.

DETAILED DESCRIPTION

[0056] The present inventors have surprisingly found that protein kinaseN beta, also referred to herein as PKN beta, is a valuable target inconnection with cancer and tumours. More particularly, the presentinventors have discovered that protein kinase N beta is a downstreamtarget of the PI-3 kinase/PTEN pathway. Still more surprisingly thepresent inventors have discovered that protein kinase N beta is linkedto tumorigenesis and metastasis.

[0057] This latter effect in particular seems to be strongly related tothe loss of suppressor function, more particularly PTEN tumoursuppressor function. As will be shown in the examples, protein kinase Nbeta is up-regulated under conditions where PTEN which is an inhibitorto the PI-3 kinase pathway, is not active. Cells in which up-regulationof protein kinase N beta occurs show an increase in metastatic behaviourand migrational behaviour. This means that inhibitors of protein kinaseN beta can be used to control metastatic and migrational behaviour ofcells and therefore provide methods of treating tumors and cancers, moreparticularly those tumors and cancers which are metastatic and the cellsof which show a metastatic and/or migrational behaviour which aregenerally referred to herein as ‘the disease as described herein’ or as‘diseased condition as described herein’.

[0058] The disease as described herein as well as the diseased conditionas described herein also includes tumorigenesis and metastasis. Thisapplies particularly to those diseases as described herein and thosediseased conditions as described herein, where the cells involved insuch diseases or diseased conditions are PTEN negative which means thatthe tumor suppressor PTEN is not active or has a reduced level ofactivity. The diseases also comprise those diseases in which the PI3-kinase pathway is in general involved.

[0059] Besides metastatic tumors in particular, diabetes falls withinthis kind of diseases and diseased condition, respectively. Accordingly,cells, particularly those which are involved in the disease or diseasedcondition as described herein and which are PTEN negative, may betreated by a drug that reduces or eliminates the activity of proteinkinase N beta in the respective cells involved. Accordingly, patientswhose tumors are characterized by a preferably hyperactivated PI3-kinase pathway, including but not limited to, either throughamplification or mutation of genes encoding components of the PI3-kinase pathway (p110, Akt) or are PTEN negative or who have cellswhich are PTEN negative, particularly if these cells are involved in thedisease as described herein or in the diseased condition as describedherein, can advantageously be treated using said drugs.

[0060] The reduction in activity may stem either from a reduction at thetranscription level or at the level of the translation, i.e. theenzymatic activity of protein kinase N beta. Without wishing to be boundby any theory, the latter aspect, i.e. modifying the activity of theprotein kinase N beta is also a result from an insight of the inventorsin relation to the characteristics of PKNbeta, namely that the enzymaticactivity of PKNbeta can also be up- and down-regulated, more preferablydown-regulated.

[0061] A further group of patients who can advantageously be treatedusing these drugs are those who suffer from cancers which have a highincidence for loss of PTEN function, especially in late stage tumors(Cantley, L. C. and Neel, B. G. (1999). New insights into tumorsuppression: PTEN suppresses tumor formation by restraining thephosphoinositide 3-kinase/AKT pathway. Proc Natl Acad Sci USA 96,4240-4245; Ali, I. U. (2000). Gatekeeper for endometrium: the PTEN tumorsuppressor gene. J Natl Cancer Inst 92, 861-863). Loss of PTENcorrelates with increased aggressive and invasive behavior of therespective tumor cells. Because of this, in preferred embodiments of thepresent invention those diagnostic agents which may also be used asanalytical tools or means in connection with the various aspects of thepresent invention, and therapeutic agents, respectively, directed toprotein kinase N beta or nucleic acids coding therefore, can be used forany tumor provided that the aforementioned prerequisite is met, namelythat PTEN correlates with increased aggressive and invasive behaviour.

[0062] This kind of drug may be designed, screened or manufactured onthe basis of the disclosure given herein, namely that protein kinase Nbeta is a downstream drug target and that protein kinase N beta is atarget for tumorigenesis and metastasis and diseases related thereto orarising therefrom.

[0063] Because of the involvement of protein kinase N beta in themechanisms as outlined above, it can also be used as a marker fordiagnosing the status of a cell or patient having in his body such kindof cells whether it will undergo metastasis and tumorigenesis,respectively. An illustration that this kind of approach works and isapplicable for that purpose is, e.g., ICAM-1. ICAM-1 is used in theprognosis of gastric cancers to undergo metastasis (Maruo Y, Gochi A,Kaihara A, Shimamura H, YamadaT, TanakaN, OritaK.Int J Cancer. 2002 Aug.1;100(4):486-490 ) where s-ICAM-1 levels were found to be elevated inpateinets with liver metasasis. In another example, osteopontin is usedas a prognostic marker for breast cancer (Rudland P S, Platt-Higgins A,El-Tanani M, De Silva Rudland S, Barraclough R, Winstanley J H, HowittR, West C R. Cancer Res. 2002 Jun. 15;62(12):3417-3427 ). In so far thepresence or the level of presence (protein or mRNA) or the level ofactivity of protein kinase N beta may be used as a marker and anycompound more or less specifically interacting with protein kinase Nbeta will therefore be an appropriate diagnostic agent.

[0064] Methods and design principles for drugs and diagnostic agentswhich in any case specifically and/or selectively interact with proteinkinase N beta will be disclosed in the following.

[0065] In the light of these findings kinase N beta proves to be asuitable downstream drug target which allows the selective modulation ofonly some aspects which are typically related to PI-3 kinase pathway,namely metastasis and migration, and a selective and specific diagnosticapproach, i.e. detection, of processes typically related to adysregulated PI 3-kinase pathway, more particularly metastasis andmigration.

[0066] The PI-3 Kinase Pathway

[0067] The PI 3-kinase pathway is characterized by a PI 3-kinaseactivity upon growth factor induction and a parallel signalling pathway.Growth factor stimulation of cells leads to activation of their cognatereceptors at the cell membrane which in turn associate with and activateintracellular signalling molecules such as PI 3-kinase. Activation of PI3-kinase (consisting of a regulatory p85 and a catalytic p110 subunit)results in activation of Akt by phosphorylation, thereby supportingcellular responses such as proliferation, survival or migration furtherdownstream. PTEN is thus a tumor suppressor which is involved in thephosphatidylinositol (PI) 3-kinase pathway and which has beenextensively studied in the past for its role in regulating cell growthand transformation (for reviews see, Stein, R. C. and Waterfield, M. D.(2000). PI3-kinase inhibition: a target for drug development? Mol MedToday 6, 347-357; Vazquez, F. and Sellers, W. R. (2000). The PTEN tumorsuppressor protein: an antagonist of phosphoinositide 3-kinasesignaling. Biochim Biophys Acta 1470, M21-35; Roymans, D. and Slegers,H. (2001). Phosphatidylinositol 3-kinases in tumor progression. Eur JBiochem 268, 487-498).

[0068] The tumor suppressor PTEN functions as a negative regulator of PI3-kinase by reversing the PI 3-kinase-catalyzed reaction and therebyensures that activation of the pathway occurs in a transient andcontrolled manner. Chronic hyperactivation of PI 3-kinase signalling iscaused by functional inactivation of PTEN. PI 3-kinase activity can beblocked by addition of the small molecule inhibitor LY294002. Theactivity and downstream responses of the signalling kinase MEK whichacts in a parallel pathway, can, for example, be inhibited by the smallmolecule inhibitor PD98059.

[0069] A chronic activation of the PI 3-kinase pathway through loss ofPTEN function is a major contributor to tumorigenesis and metastasisindicating that this tumor suppressor represents an important checkpointfor a controlled cell proliferation. PTEN knock out cells show similarcharacteristics as cells in which the PI 3-kinase pathway has beenchronically induced via activated forms of PI 3-kinase (Di Cristofano,A., Pesce, B., Cordon-Cardo, C. and Pandolfi, P. P. (1998). PTEN isessential for embryonic development and tumour suppression. Nat Genet19, 348-355. Klippel, A., Escobedo, M. A., Wachowicz, M. S., Apell, G.,Brown, T. W., Giedlin, M. A., Kavanaugh, W. M. and Williams, L. T.(1998). Activation of phosphatidylinositol 3-kinase is sufficient forcell cycle entry and promotes cellular changes characteristic ofoncogenic transformation. Mol Cell Biol 18, 5699-5711. Kobayashi, M.,Nagata, S., Iwasaki, T., Yanagihara, K., Saitoh, I., Karouji, Y., Ihara,S. and Fukui, Y. (1999). Dedifferentiation of adenocarcinomas byactivation of phosphatidylinositol 3-kinase. Proc Natl Acad Sci USA 96,4874-4879).

[0070] PTEN is involved in several pathways which are also referred toas PTEN related pathways such as the PI3K/PTEN pathway, the Akt pathway,the EGF-related autocrine loop and the mTOR pathway. A P13-kinasepathway is actually any pathway which involves PI 3-kinase, eitherdirectly or indirectly. PI 3-kinase may act either as an inhibitor or asan activator in such pathway, or it may as such be regulated by otherelements of the pathway.

[0071] Diseases and conditions involving dysregulation of the PI3-kinase pathway are well known. Any of these conditions and diseasesmay thus be addressed by the inventive methods and the drugs anddiagnostic agents the design, screening or manufacture thereof is taughtherein. For reasons of illustration but not limitation it is referred tothe following: endometrial cancer, colorectal carcinomas, gliomas,endometrial cancers, adenocarcinomas, endometrial hyperplasias, Cowden'ssyndrome, hereditary non-polyposis colorectal carcinoma, Li-Fraumene'ssyndrome, breast-ovarian cancer, prostate cancer (Ali, I. U., Journal ofthe National Cancer Institute, Vol. 92, no. 11, Jun. 7, 2000, page861-863), Bannayan-Zonana syndrome, LDD (Lhermitte-Duklos' syndrome)(Macleod, K., supra) hamartoma-macrocephaly diseases including Cowdisease (CD) and Bannayan-Ruvalcaba-Rily syndrome (BRR), mucocutaneouslesions (e.g. trichilemmonmas), macrocephaly, mental retardation,gastrointestinal harmatomas, lipomas, thyroid adenomas, fibrocysticdisease of the breast, cerebellar dysplastic gangliocytoma and breastand thyroid malignancies (Vazquez, F., Sellers, W. R., supra.)

[0072] In view of this, protein kinase N beta is a valuable downstreamdrug target of the PI 3-kinase pathway which can be addressed by drugswhich will have less side effects than other drugs directed to targetsupstream of protein kinase N beta. Insofar the present inventionprovides a drug target which is suitable for the design, screening,development and manufacture of pharmaceutically active compounds whichare more selective than those known in the art, such as, for example, LY294002. By having control over this particular fraction of effectormolecules, i.e. the protein kinase N beta and any further downstreammolecule involved in the pathway, only a very limited number of parallelbranches thereof or further upstream targets in the signalling cascadeare likely to cause unwanted effects. Therefore, the other activities ofthe PI-3 kinase/PTEN pathway related to cell cycle, DNA repair,apoptosis, glucose transport, translation will not be influenced. Also,the insulin signalling is not induced which means that the diabeticresponses or other side effects observed in connection with the use ofLY294002 are actually avoided.

[0073] LY294002 (2-(4-morpholinyl)8-phenylchromone) is one of severalchromone derivatives small molecule inhibitor developed by LillyResearch Laboratories (Indianapolis) as an inhibitor for PI-3K (Vlahoset al. 1994, J Biol Chem. 269, 5241-5248). It targets the catalyticsubunit of the PI-3K molecule, p110 and functions by competing with ADPbinding in the catalytic centre. However, LY294002 cannot distinguishbetween different isoforms of p110 (alpha, beta, gamma, delta) which aresuggested to have different cellular functions.

[0074] Protein kinase N beta is also further downstream of mTOR which isaddressed by rapamycin. mTOR (mammalian Target Of Rapamycin), also knownas Raft or FRAP, is acting downstream of PI 3-kinase to regulateprocesses such as the pp70S6 kinase dependent entry into the cell cycle.mTOR acts as a sensor for growth factor and nutrient availability tocontrol translation through activating pp70S6 kinase and initiationfactor 4E. mTOR function is inhibited by the bacterial macroliderapamycin which blocks growth of T-cells and certain tumor cells(Kuruvilla and Schreiber 1999, Chemistry & Biology 6, R129-R136).

[0075] The fact that rapamycin and its derivatives are suitable drugscurrently being used in the clinic proves that a drug target is the morehelpful and has the less side effects, the more specific it is for aparticular molecular mechanism as, e.g., demonstrated by Yu et al. (Yu,K. et al (2001) Endrocrine-RelatCanc 8, 249).

[0076] Protein kinase N beta is a member of the protein kinase C familyall of which are said to be protein-serine/threonine kinases. Typically,this kind of protein kinase comprises one regulatory and one catalyticsubunit and uses calcium ions and phospholipids as co-factors. Diacylglycerols act as activators of this kind of protein kinase family.Members of the protein kinase C family are involved in severalsignalling pathways linked to hormones or neurotransmitters. Theseprotein kinases regulate the activity of their target proteins byphosphorylation. It is known in the art that unphysiological continuedactivation of protein kinase C results in the transformed cellularphenotype that might lead to the generation of cancer.

[0077] Protein Kinase N Beta and Its Derivatives

[0078] The complete sequence of protein kinase N beta as MnRNA isavailable in databanks, e.g., under accession numbers gi 7019488 orNM_(—)013355. Using the genetic code, the particular amino acid sequencemay be deduced from this mRNA. Also, the amino acid sequence of proteinkinase N beta is available in databanks under the accession number gi7019489 or NP_(—)037487.1. It is within the present invention thatderivatives or truncated versions thereof may be used according to thepresent invention as long as the desired effects may be realised. Theextent of derivatization and truncation can thus be determined by oneskilled in the art by routine analysis.

[0079] In the context of the present invention, the term nucleic acidsequences encoding protein kinase N beta also includes nucleic acidwhich hybridise to nucleic acid sequences specified by theaforementioned accession numbers or any nucleic acid sequence which maybe derived from the aforementioned amino acid sequences. Suchhybridization is known to the one skilled in the art. Theparticularities of such hybridisation may be taken from Sambrook, J.Fritsch, E. F. and Maniats, T. (1989) Molecular Cloning: A LaboratoryManual, 2^(nd) ed. Cold Spring Harbor: Cold Spring Harbor Laboratory. Ina preferred embodiment the hybridization is a hybridization understringent conditions, for example, under the stringent conditionsspecified in Sambrook supra.

[0080] In addition, a nucleic acid coding for a protein kinase N beta isalso a nucleic acid sequence which contains sequence homologous to anyof the aforementioned nucleic acid sequences, whereby the degree ofsequence homology is preferably 75, 80, 85, 90 or 95%. Furtherreferences related to to protein kinase N beta are, among others ShibataH. et al., J. Biochem. (Tokyo) July 2001; 130 (1): 23-31; Dong, L Q,Proc Natl Acad Sci USA, 2000, May 9, 1997 (10): 5089-5094; and Oishi,K., Biochem Biophys Res Commun. 1999, Aug. 11; 261 (3): 808-814.

[0081] Homologues to human protein kinase N beta may be found, amongothers, in M. musculus, R norvegicus, A. thaliana, C. elegans, D.melanogaster and S. cerevisiae. The percent identity and length of thealigned region is 67% and 279 amino acids, 51% and 866 amino acids, 38%and 305 amino acids, 36% and 861 amino acids, 63% and 296 amino acidsand 44% and 362 amino acids, respectively, for the various speciesmentioned before. It will be acknowledged by the ones skilled in the artthat any of these or other homologues will in principle be suitable forthe practice of the present invention provided the drug or diagnosticagent generated using such homologue may still interact with the humanprotein kinase N beta or any other intended protein kinase N beta.

[0082] The human amino acid sequence may also be taken from ProtEST,accession number pir: JC7083 where the respective protein kinase N betais referred to as JC7083 protein kinase. The gene for human proteinkinase N beta is located on human chromosome number 9. cDNA sources forprotein kinase N beta are in general a number of cancers and variousfetal or embryonic tissues, more particularly, among others, stomach,adenocarcinoma, brain, breast, Burkitt's lymphoma, cervix,chondrosarcoma, colon, fetal eyes, fetal lens, fetal eye anteriorsegment, fetal optic nerve, fetal retina, fetal retina foveal, fetalmacular fetal choroid, fibrotheoma, germ line, nead neck, heart, kidney,large cell carcinoma, leiomyosarcoma metastatic chondrosarcoma, ovary,parathyroid, retinoblastoma, rhabdomyosarcoma, small cell carcinoma,squamous cell carcinoma, testis, and uterus.

[0083] From this list it is apparent that a drug (which is also referredto herein as a medicament), and the diagnostic agent, including astaging agent, i.e. antibody agent which can be used to differentiatethe status of a patient with regard to the stage of a disease from whichhe might suffer, as well as for monitoring the effectiveness of atreatment applied to a patient, respectively, to be designed, screenedor manufactured according to the technical teaching given herein may inaddition to any of the other diseases as disclosed herein and thediseased conditions as disclosed herein also be used for the treatment,prevention, diagnosis, prognosis and monitoring of these diseases or anydisease involving the specific cells, tissues or organs. These diseasesand diseased conditions are also understood in the context of theinvention to be included within the term “disease as described herein”.

[0084] Use of Protein Kinase N Beta as a Medicament

[0085] In view of the surprising findings disclosed herein, proteinkinase N beta as such may be used as a medicament for the preventionand/or treatment of the various diseases and diseased conditions asdescribed herein, and for the manufacture of a medicament for suchpurpose and for the manufacture of a diagnostic agent.

[0086] When protein kinase N beta or a fragment or derivative thereof asdefined above is used as a medicament itself, it is preferably used as acompetitor to the naturally occurring protein kinase N beta, therebypreventing the normal biological function thereof. It is particularlypreferred that the protein kinase N beta used for that purpose iscatalytically defective. This kind of protein kinase N beta may eitherbe applied to the organism and cell, respectively, or may be introducedinto the organism and respective cells by means of gene therapy.

[0087] Use of Protein Kinase N Beta as a Target

[0088] Apart from being a potential drug itself, protein kinase N betamay be used as a target against which chemical compounds which may beused as drugs or drug candidates or as diagnostic agents, are directed.Suitable chemical compounds belonging to different classes of compoundssuch as antibodies, peptides, anticalines, aptamers, spiegelmers,ribozymes, antisense oligonucleotides and siRNA as well as smallmolecules may be used. The compounds are designed, selected, screenedgenerated and/or manufactured by either using protein kinase N betaitself as a physical or chemical entity, or information related toprotein kinase N beta.

[0089] In the design, selection, screening, generation and/ormanufacturing process of said classes of compounds protein kinase N betawill also be referred to as the target which is used in the processrather than in the final application of the respective compound to apatient in need thereof. In the processes which provide the variousclasses of compounds, either the protein protein kinase N beta, alsoreferred to herein as protein kinase N beta or a nucleic acid coding forprotein kinase N beta may be used. The term protein kinase N beta asused herein comprises any fragment or derivative of protein kinase Nbeta which allows the design, selection, screening, generation and/ormanufacture of said classes of compounds of the respective class(es) ofcompounds which in turn are/is upon their/its application as amedicament or as a diagnostic agent active as such.

[0090] The term nucleic acid coding for protein kinase N beta as usedherein shall comprise any nucleic acid which contains a nucleic acidwhich codes for protein kinase N beta as defined above, or a partthereof. A part of a nucleic acid coding for protein kinase N beta isregarded as such as long as it is still suitable for the design,selection, screening, generation and/or manufacture of said classes ofcompounds which in turn are/is upon their/its application as amedicament or as a diagnostic agent active as such. The nucleic acidcoding for protein kinase beta N may be genomic nucleic acid, hnRNA,mRNA, cDNA or part of each thereof.

[0091] As outlined above it is within the present invention that apartfrom protein kinase N beta or a part or derivative thereof or a nucleicacid sequence therefore, as described herein, also other means orcompounds may be used in order to create or to suppress the effectsarising from protein kinase N beta or the nucleic acid coding proteinkinase N beta. Such means may be determined or selected in a screeningmethod. In such screening method a first step is to provide one orseveral so called candidate compounds. Candidate compounds as usedherein are compounds the suitability of which is to be tested in a testsystem for treating or alleviating the various diseases as describedherein and diseased conditions as described herein or to be used as adiagnostic means or agent for this kind of diseases and diseasedconditions.

[0092] If a candidate compound shows a respective effect in a testsystem said candidate compound is a suitable means or agent for thetreatment of said diseases and diseased conditions and, in principle, aswell a suitable diagnostic agent for said diseases and diseasedconditions. In a second step the candidate compound is contacted with aprotein kinase N beta expression system or a protein kinase N beta geneproduct, preferably a respective gene expression product, such as ahnRNA or MRNA, or a protein kinase N beta activity system or a proteinkinase N beta. The protein kinase N beta activity system is alsoreferred to herein as and/or is preferably also active in the meaning ofa system detecting the activity of protein kinase N beta.

[0093] The protein kinase N beta screening methodology described hereinalso is useful to eliminate non-functional or inactive compounds fromfurther consideration. Thus protein kinase N beta activity can bemeasured in a first sample obtained from a subject or test system,generating a pre-treatment level, followed by administering a test agentto the subject or test system and measuring protein kinase N betaactivity in a second sample from the subject or test system at a timefollowing administration of the test agent, thereby generating data fora test level. The pre-treatment level can be compared to the test level,and data showing no decrease in the test level relative to thepre-treatment level indicates that the test agent is not effective inthe subject, and the test agent may be eliminated from furtherevaluation or study. Conversely, a change in values can indicate thatthe test agent is suitable for use as a PKN beta inhibitor or forfurther study.

[0094] A protein kinase N beta expression system as that term is usedherein is basically an expression system that shows or displays theexpression of protein kinase N beta, whereby the extent or level ofexpression may be changed. Preferably, a protein kinase N beta activitysystem is essentially an expression system whereby the activity orcondition of activity is measured rather than the expression of proteinkinase N beta. Alternatively, a protein kinase activity system is aprotein kinase N beta the activity of which can be measured, or a systemproviding or comprising protein kinase N beta.

[0095] Detection and Inhibition of Protein Kinase N Beta Activity

[0096] In any of these systems it is determined whether under theinfluence of a candidate compound the activity of protein kinase N betaor of the nucleic acid coding protein kinase N beta is different fromthe situation without the candidate compound. Regardless whether theparticular system is either an expression system or an activity system,it is within the scope of the present invention that either an increaseor a decrease of the activity and expression, respectively, may occurand be measured. Typically, the expression system and/or activity systemis an in vitro reaction, such as a cell extract or a fraction of thecell extract such as a nuclear extract. A protein kinase N betaexpression system as used herein may also be a cell, preferably a cellof a tissue or organ involved in the diseases as described herein anddiseased conditions as described herein.

[0097] Whether there is an increase or decrease in the activity systemor expression system may be determined at each level of the expression,for example by measuring the increase or decrease of the amount ofnucleic acid coding for protein kinase N beta, more particularly mRNA,or the increase or decrease of protein kinase N beta polypeptideexpressed under the influence of the candidate compound. The techniquesrequired for such measurements, more particularly the quantitativemeasurement of these kinds of changes, such as for the MRNA or theprotein are known to the one skilled in the art. Also known to the oneskilled in the art are methods to determine the amount of or content ofprotein kinase N beta, e.g. by detection using appropriate antibodies.Antibodies may be generated as known to the one skilled in the art anddescribed, e.g. by Harlow, E., and Lane, D., “Antibodies: A LaboratoryManual,” Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,(1988).Suitable antibodies may also be generated by other well known methods,for example, by phage display selection from libraries of antibodies.

[0098] In case of a protein kinase N beta expression system an increaseor decrease of the activity of protein kinase N beta may be determined,preferably in a functional assay.

[0099] Contacting the candidate compound and the expression system andactivity system, respectively, usually is performed by adding an aqueoussolution of the candidate compound to a respective reaction system whichis generally referred to herein as the test system. Besides aqueoussolutions, suspensions or solutions of the candidate compound in organicsolvents or in mixtures of organic and aqueous solvents may be used. Theaqueous solution is preferably a buffer solution.

[0100] Preferably, in each run using the expression system and activitysystem, respectively, only a single candidate compound is used. However,it is also within the present invention that several of this kind oftests are performed in parallel in a high throughput system usingmethods known in the art.

[0101] A further step in the method according to the present inventionresides in determining whether under the influence of the candidatecompound the expression or activity of the expression system andactivity system, respectively, in relation to protein kinase N beta or anucleic acid coding therefore is changed. Typically this is done bycomparing the system's reaction upon addition of the candidate compoundrelative to the one without addition of the candidate compound.Preferably, the candidate compound is a member of a library ofcompounds.

[0102] Basically any library of compounds is suitable for the purpose ofthis invention regardless of the class of compounds. Suitable librariesof compounds are, among others, libraries composed of small molecules,of peptides, proteins, antibodies, anticalines and functional nucleicacids. The latter compounds may be generated as known to the one skilledin the art and outlined herein.

[0103] Antibodies

[0104] The manufacture of an antibody specific for the protein ofprotein kinase N beta or for the nucleic acid coding for protein kinaseN beta, is known to the one skilled in the art and, for example,described in Harlow, E., and Lane, D., “Antibodies: A LaboratoryManual,” Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,(1988).Monoclonal antibodies may be used in connection with the presentinvention which may be manufactured according to the protocol of Cesarand Milstein and further developments based thereon, for example byselection from antibody libraries by, for example, phage display. Seefor example, U.S. Pat. No. 5,969,108, the disclosure of which is herebyincorporated by reference in its entirety. Antibodies as used herein,include, but are not limited to, complete antibodies, antibody fragmentsor derivatives such as Fab fragments, Fc fragments and single-strandedantibodies, as long as they are suitable and capable of binding toprotein kinase N beta. Apart from monoclonal antibodies also polyclonalantibodies may be used and/or generated. The generation of polyclonalantibodies is also known to the one skilled in the art and, for example,described in Harlow and Lane supra. Preferably, the antibodies used fortherapeutic purposes are humanized or human antibodies as defined above.

[0105] The antibodies which may be used according to the presentinvention may have one or several markers or labels. Such markers orlabels may be useful to detect the antibody either in its diagnosticapplication or its therapeutic application. Preferably the markers andlabels are selected from the group comprising avidine, streptavidine,biotin, gold and fluorescein and used, e.g., in ELISA methods. These andfurther markers as well as methods are, e.g. described in Harlow andLane, supra.

[0106] It is also within the present invention that the label or markerexhibits an additional function apart from detection, such asinteraction with other molecules. Such interaction may be, e.g.,specific interaction with other compounds. These other compounds mayeither be those inherent to the system where the antibody is used suchas the human or animal body or the sample which is analysed by using therespective antibody. Appropriate markers may, for example, be biotin orfluorescein with the specific interaction partners thereof such asavidin and streptavidin and the like being present on the respectivecompound or structure to interact with the thus marked or labelledantibody.

[0107] Peptides

[0108] A further class of medicaments as well as diagnostic agents whichmay be generated using the protein of protein kinase N beta or thenucleic acid coding for protein kinase beta, are peptides which bindthereto. Such peptides may be generated by using methods according tothe state of the art such as phage display. Basically, a library ofpeptides is generated and displayed on the surface of phage, and thedisplayed library is contacted with the target molecule, in the presentcase, for example, the protein kinase N beta. Those peptides binding tothe target molecule are subsequently removed, preferably as a complexwith the target molecule, from the respective reaction. It is known tothe one skilled in the art that the binding characteristics, at least toa certain extend, depend on the particularly realized experimentalset-up such as the salt concentration and the like. After separatingthose peptides binding to the target molecule with a higher affinity ora bigger force, from the non-binding members of the library, andoptionally also after removal of the target molecule from the complex oftarget molecule and peptide, the respective peptide(s) may subsequentlybe characterized.

[0109] Prior to the characterisation optionally an amplification step isrealized such as, e.g. by propagating the peptide coding phages. Thecharacterization preferably comprises the sequencing of the targetbinding peptides. Basically, the peptides are not limited in theirlengths, however, preferably peptides having a lengths from about 8 to20 amino acids are preferably obtained in the respective methods. Thesize of the libraries may be about 10² to 10¹⁸, preferably 10⁸ to 10¹⁵different peptides, however, is not limited thereto.

[0110] Anticalines

[0111] A particular form of target binding polypeptides are theso-called “anticalines” which are, among others, described in Germanpatent application DE 197 42 706, the disclosure of which is herebyincorporated by reference.

[0112] According to the present invention the protein of protein kinaseN beta as well as the nucleic acid coding for protein kinase N beta maybe used as the target for the manufacture or development of a medicamentfor the treatment of the diseases described herein and of the diseasedconditions described herein, as well as for the manufacture and/ordevelopment of means for the diagnosis of said diseases and saidconditions, in a screening process, whereby in the screening processsmall molecules or libraries of small molecules are used. This screeningcomprises the step of contacting the target molecule with a single smallmolecule or a variety (such as a library) of small molecules at the sametime or subsequently, preferably those from the library as specifiedabove, and identifying those small molecules or members of the librarywhich bind to the target molecules which, if screened in connection withother small molecules may be separated from the non-binding ornon-interacting small molecules.

[0113] The binding and non-binding may strongly be influenced by theparticular experimental set-up. In modifying the stringency of thereaction parameters it is possible to vary the degree of binding andnon-binding which allows a fine tuning of this screening process.Preferably, after the identification of one or several small moleculeswhich specifically interact with the target molecule, this smallmolecule may be further characterized. This further characterisationmay, for example, reside in the identification of the small molecule anddetermination of its molecule structure and further physical, chemical,biological and/or medical characteristics. Preferably, the naturalcompounds have a molecular weight of about 100 to 1000 Da. Alsopreferably, small molecules are those which comply with the Lepinskyrules of five known to the ones skilled in the art. Alternatively, smallmolecules may also be defined such that they aresynthetic-small-molecules, preferably arising from combinatorialchemistry, in contrast to natural products which preferably arenon-synthetic. However, it is to be noted that these definitions areonly subsidiary to the general understanding of the respective terms inthe art. Like all kinases, protein kinase N beta contains an ATP-bindingsite and drugs that are known to bind to such sites are thereforesuitable candidate compounds for inhibiting protein kinase N beta.Examples if suitable compounds include, but are not limited to, Y-27632,Ro-3 1-8220, and HA 1077, all of which are available from Calbiochem (LaJolla, Calif.).

[0114] Aptamers and Speigelmers

[0115] It is also within the present invention to use the protein kinaseN beta and/or a nucleic acid coding for protein kinase N beta as atarget molecule for the manufacture or selection of aptamers andspiegelmers which may then be used directly or indirectly either asmedicament or as diagnostic agents.

[0116] Aptamers are D-nucleic acids which are either single stranded ordouble stranded and which specifically interact with a target molecule.The manufacture or selection of aptamers is, e.g., described in Europeanpatent EPO 533 838, the specification of which is hereby incorporated byreference in its entirety. Basically the following steps are realized.First, a mixture of nucleic acids, i.e. potential aptamers, is providedwhereby each nucleic acid typically comprises a segment of several,preferably at least eight, subsequent randomised nucleotides. Thismixture is subsequently contacted with the target molecule whereby thenucleic acid(s) bind to the target molecule, such as based on anincreased affinity towards the target or with a bigger force thereto,compared to the candidate mixture. The binding nucleic acid(s) are/issubsequently separated from the remainder of the mixture. Optionally,the nucleic acid(s) thus obtained is amplified using, e.g. a polymerasechain reaction. These steps may be repeated several times giving at theend a mixture having an increased ratio of nucleic acids specificallybinding to the target from which the final binding nucleic acid is thenoptionally selected. These specifically binding nucleic acid(s) arereferred to aptamers.

[0117] It is apparent that at any stage of the method for the generationor identification of the aptamers samples of the mixture of individualnucleic acids may be taken to determine the sequence thereof usingstandard techniques. It is within the present invention that theaptamers may be stabilized -such as, e.g., by introducing definedchemical groups which are known to the one skilled in the art ofgenerating aptamers. Such modification may for example reside in theintroduction of an amino group at the 2′-position of the sugar moiety ofthe nucleotides. Aptamers are currently used as therapeutical agents.

[0118] However, it is also within the present invention that the thusselected or generated aptamers may be used for target validation and/oras lead substance for the development of medicaments, preferably ofmedicaments based on small molecules. This is actually done by acompetition assay whereby the specific interaction between the targetmolecule and the aptamer is inhibited by a candidate drug whereby uponreplacement of the aptamer from the complex of target and aptamer it maybe assumed that the respective drug candidate allows a specificinhibition of the interaction between target and aptamer, and if theinteraction is specific, said candidate drug will, at least inprinciple, be suitable to block the target and thus decrease itsbiological availability or activity in a respective system comprisingsuch target. The small molecule thus obtained may then be subject tofurther derivatisation and modification to optimise its physical,chemical, biological and/or medical characteristics such as toxicity,specificity, biodegradability and bioavailability.

[0119] The generation or manufacture of spiegelmers which may be used orgenerated according to the present invention using protein kinase N betaor a nucleic acid coding for protein kinase N beta, is based on asimilar principle. The manufacture of spiegelmers is described in theinternational patent application WO 98/08856. Spiegelmers are L-nucleicacids, which means that they are composed of L-nucleotides rather thanaptamers which are composed of D-nucleotides as aptamers are.Spiegelmers are characterized by the fact that they have a very highstability in biological system and, comparable to aptamers, specificallyinteract with the target molecule against which they are directed. Inthe purpose of generating spiegelmers, a heterogonous population ofD-nucleic acids is created and this population is contacted with theoptical antipode of the target molecule, in the present case for examplewith the D-enantiomer of the naturally occurring L-enantiomer of theprotein kinase N beta. Subsequently, those D-nucleic acids are separatedwhich do not interact with the optical antipode of the target molecule.However, those D-nucleic acids interacting with the optical antipode ofthe target molecule are separated, optionally determined and/orsequenced and subsequently the corresponding L-nucleic acids aresynthesized based on the nucleic acid sequence information obtained fromthe D-nucleic acids. These L-nucleic acids which are identical in termsof sequence with the aforementioned D-nucleic acids interacting with theoptical antipode of the target molecule, will specifically interact withthe naturally occurring target molecule rather than with the opticalantipode thereof. Similar to the method for the generation of aptamersit is also possible to repeat the various steps several times and thusto enrich those nucleic acids specifically interacting with the opticalantipode of the target molecule.

[0120] Ribozymes, Antisense Oligonucleotides and siRNA.

[0121] A further class of compounds which may be manufactured orgenerating based on protein kinase N beta or a nucleic acid coding forprotein kinase beta, as the target molecule as disclosed herein, areribozymes, antisense oligonucleotides and siRNA.

[0122] It is a common feature of all of the aforementioned nucleic acidsthat they do not interact with the target molecule at the level of thetranslation product which is in the present case the protein kinase Nbeta, but rather interact with the transcription product, i.e. thenucleic acid coding for protein kinase beta such as the genomic nucleicacid or any nucleic acid derived therefrom such as the correspondinghnRNA, cDNA and MRNA, respectively. Insofar, the target molecule of theaforementioned class of compounds is preferably the mRNA of proteinkinase N beta.

[0123] Ribozymes

[0124] Ribozymes are catalytically active nucleic acids which preferablyconsist of RNA which basically comprises two moieties. The first moietyshows a catalytic activity whereas the second moiety is responsible forthe specific interaction with the target nucleic acid, in the presentcase the nucleic acid coding for protein kinase N beta. Upon interactionbetween the target nucleic acid and the second moiety of the ribozyme,typically by hybridisation and Watson-Crick base pairing of essentiallycomplementary stretches of bases on the two hybridising strands, thecatalytically active moiety may become active which means that itcatalyses, either intramolecularly or intermolecularly, the targetnucleic acid in case the catalytic activity of the ribozyme is aphosphodiesterase activity. Subsequently, there may be a furtherdegradation of the target nucleic acid which in the end results in thedegradation of the target nucleic acid as well as the protein derivedfrom the said target nucleic acid which in the present case is proteinkinase N beta due to a lack of newly synthesized protein kinase N betaand a turn-over of prior existing protein kinase N beta. Ribozymes,their use and design principles are known to the one skilled in the art,and, for example described in Doherty and Doudna (Ribozyme structuresand mechanism. Annu Rev. Biophys. Biomolstruct. 2001; 30 :457-75) andLewin and Hauswirth (Ribozyme Gene Therapy: Applications for molecularmedicine. 20017: 221-8).

[0125] Antisense Oligonucleotides

[0126] The use of antisense oligonucleotides for the manufacture of amedicament and as a diagnostic agent, respectively, is based on asimilar mode of action. Basically, antisense oligonucleotides hybridisebased on base complementarity, with a target RNA, preferably with amRNA, thereby activating RNase H. RNase H is activated by bothphosphodiester and phosphorothioate-coupled DNA. Phosphodiester-coupledDNA, however, is rapidly degraded by cellular nucleases butphosphorothioate-coupled DNA is more stable. These resistant,non-naturally occurring DNA derivatives do not inhibit RNase H uponhybridisation with RNA. In other words, antisense polynucleotides areonly effective as DNA RNA hybrids complexes. Examples for this kind ofantisense oligonucleotides are described, among others, in U.S. Pat.Nos. 5,849,902 and 5,989,912. In other words, based on the nucleic acidsequence of the target molecule which in the present case is the nucleicacid coding for protein kinase N beta, either from the target proteinfrom which a respective nucleic acid sequence may in principle bededuced, or by knowing the nucleic acid sequence as such, particularlythe mRNA, suitable antisense oligonucleotides may be designed base onthe principle of base complementarity.

[0127] Particularly preferred are antisense-oligonucleotides which havea short stretch of phosphorothioate DNA (3 to 9 bases). A minimum of 3DNA bases is required for activation of bacterial RNase H and a minimumof 5 bases is required for mammalian RNase H activation. In thesechimeric oligonucleotides there is a central region that forms asubstrate for RNase H that is flanked by hybridising “arms” comprised ofmodified nucleotides that do not form substrates for RNase H. Thehybridising arms of the chimeric oligonucleotides may be modified suchas by 2′-O-methyl or 2′-fluoro. Alternative approaches usedmethylphosphonate or phosphoramidate linkages in said arms. Furtherembodiments of the antisense oligonucleotide useful in the practice ofthe present invention are P-methoxyoligonucleotides, partialP-methoxyoligodeoxyribonucleotides or P-methoxyoligonucleotides.

[0128] Of particular relevance and usefulness for the present inventionare those antisense oligonucleotides as more particularly described inthe above two mentioned U.S. patents. These oligonucleotides contain nonaturally occurring 5′→3′-linked nucleotides. Rather theoligonucleotides have two types of nucleotides:2′-deoxyphosphorothioate, which activate RNase H, and 2′-modifiednucleotides, which do not. The linkages between the 2′-modifiednucleotides can be phosphodiesters, phosphorothioate orP-ethoxyphosphodiester. Activation of RNase H is accomplished by acontiguous RNase H-activating region, which contains between 3 and 52′-deoxyphosphorothioate nucleotides to activate bacterial RNase H andbetween 5 and 10 2′-deoxyphosphorothioate nucleotides to activateeucaryotic and, particularly, mammalian RNase H. Protection fromdegradation is accomplished by making the 5′ and 3′ terminal baseshighly nuclease resistant and, optionally, by placing a 3′ terminalblocking group.

[0129] More particularly, the antisense oligonucleotide comprises a 5′terminus and a 3′ terminus; and from 11 to 59 5′→3′-linked nucleotidesindependently selected from the group consisting of 2′-modifiedphosphodiester nucleotides and 2′-modified P-alkyloxyphosphotriesternucleotides; and wherein the 5′-terminal nucleoside is attached to anRNase H-activating region of between three and ten contiguousphosphorothioate-linked deoxyribonucleotides, and wherein the3′-terminus of said oligonucleotide is selected from the groupconsisting of an inverted deoxyribonucleotide, a contiguous stretch ofone to three phosphorothioate 2′-modified ribonucleotides, a biotingroup and a P-alkyloxyphosphotriester nucleotide.

[0130] Also an antisense oligonucleotide may be used wherein not the 5′terminal nucleoside is attached to an RNase H-activating region but the3′ terminal nucleoside as specified above. Also, the 5′ terminus isselected from the particular group rather than the 3′ terminus of saidoligonucleotide.

[0131] Suitable and useful antisense oligonucleotides are also thosecomprising a 5′ terminal RNase H activating region and having between 5and 10 contiguous deoxyphosphorothioate nucleotides; between 11 to 59contiguous 5′→3′-linked 2′-methoxyribonucleotides; and an exonucleaseblocking group present at the 3′ end of the oligonucleotide that isdrawn from the group consisting of a non-5 ′-3′-phosphodiester-linkednucleotide, from one to three contiguous 5′-3′-linked modifiednucleotides and a non-nucleotide chemical blocking group.

[0132] Two classes of particularly preferred antisense oligonucleotidescan be characterized as follows:

[0133] The first class of antisense oligonucleotides, also referred toherein as second generation of antisense oligonucleotides, comprises atotal of 23 nucleotides comprising in 5′→3′ direction a stretch of seven2′-O-methylribonucleotides, a stretch of nine 2′-deoxyribonucleotides, astretch of six 2′-O-methylribonucleotides and a 3′-terminal2′-deoxyribonucleotide. From the first group of seven2′-O-methylribonucleotides the first four are phosphorothioate linked,whereas the subsequent four 2′-O-methylribonucleotides arephosphodiester linked. Also, there is a phosphodiester linkage betweenthe last, i.e. the most 3 ′-terminal end of the2′-O-methylribonucleotides and the first nucleotide of the stretchconsisting of nine 2′-deoxyribonucleotides. All of the2′-deoxyribonucleotides are phosphorothioate linked. A phosphorothioatelinkage is also present between the last, i.e. the most 3 ′-terminal 2′-deoxynucleotide, and the first 2 ′-O-methylribonucleotide of thesubsequent stretch consisting of six 2′-O-methylribonucleotides. Fromthis group of six 2′-O-methylribonucleotides the first four of them,again in 5′→3′ direction, are phosphodiester linked, whereas the lastthree of them, corresponding to positions 20 to 22 are phosphorothioatelinked. The last, i.e. terminal 3′-terminal 2′-deoxynucleotide is linkedto the last, i.e. most 3′-terminal 2′-O-methylribonucleotide through aphosphorothioate linkage.

[0134] This first class may also be described by reference to thefollowing schematic structure: RRRnnnnNNNNNNNNNnnnRRRN. Hereby, Rindicates phosphorothioate linked 2′-O-methyl ribonucleotides (A, G, U,C); n stands for 2′-O-methyl ribonucleotides (A, G, U, C); N representsphosphorothioate linked deoxyribonucleotides (A, G, T, C).

[0135] The second class of particularly preferred antisenseoligonucleotides, also referred to herein as third generation (of)antisense oligonucleotides or GeneBlocs, also comprises a total of 17 to23 nucleotides with the following basic structure (in 5′→3′ direction).

[0136] At the 5′-terminal end there is an inverted abasic nucleotidewhich is a structure suitable to confer resistance against exonucleaseactivity and, e.g., described in WO 99/54459. This inverted abasic islinked to a stretch of five to seven 2′-O-methylribonucleotides whichare phosphodiester linked. Following this stretch of five to seven2′-O-methylribonucleotides there is a stretch of seven to nine2′-deoxyribonucleotides all of which are phosphorothioate linked. Thelinkage between the last, i.e. the most 3′-terminal2′-O-methylribonucleotide and the first 2′-deoxynucleotide of the2′-deoxynucleotide comprising stretch occurs via a phosphodiesterlinkage. Adjacent to the stretch of seven to nine 2′-deoxynucleotides astretch consistent of five to seven 2′-O-methylribonucleotides S isconnected. The last 2′-deoxynucleotide is linked to the first2′-O-methylribonucleotide of the latter mentioned stretch consisting offive to seven 2′-O-methylribonucleotides occurs via a phosphorothioatelinkage. The stretch of five to seven 2′-O-methylribonucleotides arephosphodiester linked. At the 3′-terminal end of the second stretch offive to seven 2′-O-methylribonucleotide another inverted abasic isattached.

[0137] This second class may also be described by reference to thefollowing schematic structure: (GeneBlocs representing the 3rdgeneration of antisense oligonucleotides have also the followingschematic structure:) cap-(n_(p))_(x)(N_(s))_(y)(n_(p))_(z)-cap orcap-nnnnnnnNNNNNNNNnnnnnnn-cap. Hereby, cap represents inverted deoxyabasics or similar modifications at both ends; n stands for 2′-O-methylribonucleotides (A, G, U, C); N represents phosphorothioate-linkeddeoxyribonucleotides (A, G, T, C); x represents an integer from 5 to 7;y represents an integer from 7 to 9; and z represents an integer from 5to 7.

[0138] It is to be noted that the integers x, y and z may be chosenindependently from each other although it is preferred that x and z arethe same in a given antisense oligonucleotide. Accordingly, thefollowing basic designs or structures of the antisense oligonucleotidesof the third generation can be as follows:cap-(n_(p))₅(N_(s))₇(n_(p))₅-cap, cap-(n_(p))₆(N_(s))₇(n_(p))₅-cap,cap-(n_(p))₇(N_(s))₇(n_(p))₅-cap, cap-(n_(p))₅(N_(s))₈(n_(p))₅-cap,cap-(n_(p))₆(N_(s))₈(n_(p))₅-cap, cap-(n_(p))₇(N_(s))₈(n_(p))₅-cap,cap-(n_(p))₅(N_(s))₉(n_(p))s-cap, cap-(n_(p))₆(NS)₉(n_(p))S-cap,cap-(n_(p))₇(NS)g(n_(p))₅-cap, cap-(n_(p))₅(NS)₇(n_(p))₆-cap,cap-(n_(p))₆(N_(s))₇(n_(p))₆-cap, cap-(n_(p))₇(N_(s))₇(n_(p))₆-cap,cap-(n_(p))₅(N_(s))₈(n_(p))₆-cap, cap-(n_(p))₆(N_(s))₈(n_(p))₆-cap,cap-(n_(p))₇(N_(s))₈(n_(p))₆-cap, cap-(n_(p))₅(N_(s))₉(n_(p))₆-cap,cap-(n_(p))₆(N_(s))₉(n_(p))₆-cap, cap-(n_(p))₇(N_(s))₉(n_(p))₆-cap,cap-(n_(p))₅(N_(s))₇(n_(p))₇-cap, cap-(n_(p))₆(N_(s))₇(n_(p))₇-cap,cap-(n_(p))₇(N_(s))₇(n_(p))₇cap, cap-(n_(p))₅(NS)s(n_(p))₇-cap,cap-(n_(p))₆(N_(s))₈(n_(p))₇-cap, cap-(n_(p))₇(N_(s))g(n_(p))₇-cap,cap-(n_(p))₅(N_(s))₉(n_(p))₇-cap, cap-(n_(p))₆(N₅)₉(n_(p))₇-cap andcap-(n_(p))₇(NS)₉(n_(p))₇-cap.

[0139] siRNA Molecules and RNAi

[0140] A further class of compounds which may be generated based on thetechnical teaching given herein and which may be used as medicamentsand/or diagnostic agents are small interfering RNA (siRNA) directed tothe nucleic acid, preferably MRNA, coding for protein kinase N beta.siRNA is a double stranded RNA having typically a length of about 21 toabout 23 nucleotides. The sequence of one of the two RNA strandscorresponds to the sequence of the target nucleic acid such as thenucleic acid coding for protein kinase N beta, to be degraded. In otherwords, knowing the nucleic acid sequence of the target molecule, in thepresent case protein kinase N beta, preferably the MRNA sequence, adouble stranded RNA may be designed with one of the two strands beingcomplementary to said, e.g. MRNA of protein kinase N beta and, uponapplication of said siRNA to a system containing the gene, genomic DNA,hnRNA or MRNA coding for protein kinase N beta, the respective targetnucleic acid will be degraded and thus the level of the respectiveprotein be reduced. The basic principles of designing, constructing andusing said siRNA as medicament and diagnostic agent, respectively, is,among others, described in international patent applications WO 00/44895and WO 01/75164.

[0141] Based on the aforementioned design principles, it is possible togenerate such siRNA, antisense oligonucleotide and ribozyme,respectively, once the nucleic acid sequence coding for protein kinase Nbeta is known. This is also true for precursor molecules of nucleic acidsuch as hnRNA, cDNA and the like, including genomic nucleic acid. Ofcourse, also knowing the respective antisense strand may allow thedesign of such nucleic acid based compounds given the basic principle ofbase pair complementarity, preferably based on Watson-Crick basepairing. Accordingly, a further aspect of the present invention isrelated to specific siRNAs, ribozymes and antisense nucleotides whichare directed against or specific for protein kinase N-beta. In thefollowing, this is further illustrated by siRNA, however, this appliesto antisense oligonucleotides and ribozymes as well, as will beacknowledged by the ones skilled in the art.

[0142] Such siRNA comprises preferably a length of from 15 to 25nucleotides, whereby this means actually any length comprising 15, 16,17, 18, 20, 21, 22, 23, 24 or 25 nucleotides. In further embodiments,the siRNA may even exhibit more nucleotides. According the designprinciples well known in the art, respective siRNA can be generated.Accordingly, the siRNA claimed herein comprises a stretch of preferablyany nucleotide length from 15 to 25 consecutive nucleotides which iseither at least partially complementary to the sense or to the antisensestrand encoding PKN-beta, and a second ribonucleotide strand which is atleast partially complementary to the first one and thus to the antisensestrand and sense strand respectively, encoding protein kinase N-beta.Any design principle known in the art of generation or manufacture ofsiRNA may be applied to this kind of duplex structure. The siRNA spacedisclosed herein comprises siRNA molecules the antisense strand of whichstarts with a nucleotides which corresponds to nucleotide no. 1 of aPKN-beta encoding sequence as specified above. Further such siRNAmolecules start with a nucleotide which corresponds to-nucleotide no 2of a PKN-beta encoding sequence as specified above, and so on. This kindof scanning over the PKN-beta encoding sequence is repeated so as toprovide all possible siRNA molecules which can be directed againstPKN-beta. The length of any of the siRNA molecules thus generated may beany length suitable for siRNA, more particularly any length as specifiedabove. Preferably, the various siRNA molecule of the siRNA moleculespace disclosed herein, overlap except the most 5′terminal nucleotide ofthe antisense strand or sense strand. It is obvious that the thusobtained antisense sequences have to complemented through base pairingso as to form the at least partially double-stranded structure requiredfor a functionally active siRNA

[0143] Pharmaceutical and Diagnostic Compositions

[0144] Based on the mode of action of the aforementioned classes ofcompounds, such as antibodies, peptides, anticalines, aptamers,spiegelmers, ribozymes, antisense oligonucleotides as well as siRNA, itis thus also within the present invention to use any of these compoundstargeting protein kinase N beta and the nucleic acid coding therefore,respectively, for the manufacture of a medicament or a diagnostic agentfor any of the diseases as described herein and any of the diseasedconditions described herein. Furthermore, these agents may be used tomonitor the progression of said diseases and diseased conditions and thesuccess of any therapy applied, respectively.

[0145] The various classes of compounds designed according to thepresent invention such as antibodies, peptides, anticalines, smallmolecules, aptamers, spiegelmers, ribozymes, antisense oligonucleotidesand siRNA may also be contained in a pharmaceutical composition.Preferably such pharmaceutical composition is used for the treatment ofthe diseases as described herein or the diseased conditions describedherein. The pharmaceutical composition may comprise in an embodiment oneor several of the aforementioned classes of compounds and/or one or moremembers of a single class, and optionally a further pharmaceuticalactive compound, and a pharmaceutically acceptable carrier. Such carriermay be either liquid or solid, for example a solution, a buffer, analcoholic solution or the like. Suitable solid carriers are, amongothers, starch and the like. It is known to the one skilled in the artto provide respective formulations for the various compounds accordingto the aforementioned classes of compounds in order to realize theparticular route of administrations such as oral, parenteral,subcutaneous, intravenous, intramuscular and the like.

[0146] The various compounds of the different classes of compounds asmentioned above, may also be, either alone or in combination, subject toor contained in a kit. Such kit comprises apart from the respectivecompound(s) additionally one or several further elements or compoundswhereby the elements are selected from the group comprising buffers,negative controls, positive controls and instructions on the use of thevarious compounds. Preferably, the various compounds are present ineither dry or liquid form, preferably as a unit dosage for a singleadministration each. The kit may particularly be used for the therapy,diagnosis or monitoring of the progress of the disease or appliedtherapies in relation to the diseases and diseased conditions asdescribed herein.

[0147] The invention is further exemplified by the following examples,which are not limiting of the scope of the invention.

EXAMPLE 1

[0148] Materials and Methods

[0149] Cell Culture

[0150] Human prostate carcinoma PC-3 cells were obtained from theAmerican Type Culture Collection (ATCC). Cells were cultured in FI2KNutrient Mixture (Kaighn's modification) containing, 10% fetal calfserum (CS), gentamycin (50 μg/ml) and amphotericin (50 ng/ml).

[0151] Transfections were carried out in 96 well or 10-cm plates (at 30%to 50% confluency) by using various cationic lipids such asOligofectarnine, Lipofectamine (Life Technologies), Argfectin50 orProfectin50 (Atugen/GOT Berlin, Germany), or FuGene 6 (Roche) accordingto the manufacturer's instructions. GeneBlocs were transfected by addingpre-formed 5× concentrated complex of GeneBloc and lipid in serum-freemedium to cells in complete medium. The total transfection volume was100 μl for cells plated in 96 wells and 10 ml for cells in 10 cm plates.The final lipid concentration was 0.8 to 1.2 μg/ml depending on celldensity; the GeneBloc concentration is indicated in each experiment.

[0152] Cultivated cells were trypsinized and harvested followingstopping the trypsin effect by medium. The cells were washed (PBS;Centrifugation 5 min/1.000 rpm) and, finally, the pellet was resuspendedat a concentration that depended on the cell number and volume to beinoculated.

[0153] Determination of the Relative Amounts of RNA Levels by TaqmanAnalysis.

[0154] RNA from cells transfected in 96-wells was isolated and purifiedusing the Invisorb RNA HTS 96 kit (InVitek GmbH, Berlin). Inhibition ofPKN beta mRNA expression was detected by real time RT-PCR (Taqman)analysis using 300 nM PKNbeta 5′ primer, 300 nM PKNbeta 3′ primer and100 nM of the PKNbeta Taqman probe Fam-Tainra labelled. The reaction wascarried out in 50 gl and assayed on the ABI PRISM 7700 Sequence detector(Applied Biosystems) according to the manufacturer's instructions underthe following conditions: 48° C. for 30 min, 95° C. for 10 min, followedby 40 cycles of 15 sec at 95° C. and 1 min at 60° C.

[0155] In Vitro Growth on Matrigel Matrix.

[0156] PC3 cells were tretated with 5 μM LY294002 or DMSO when seeded onMatrigel. If cells were trnasfected previous to seeding cells weretransfected with GeneBloc and trypsinized 48 h post transfection. Thecells were washed in medium and seeded into duplicate 24-wells (100.000cells per well) pre-coated with 250 μl matrigel basement membrane matrix(Becton Dickinson). After incubation for 24 to 72 h photographs weretaken at 5× magnification with an Axiocam camera attached to an AxiovertS100 microscope (Zeiss).

[0157] Affymetrix

[0158] Total RNA from cells grown on Matrigel was prepared using TotallyRNA kit (AMBION) following manufacturers protocol. In the final stepprecipitated total RNA was resuspended in Invisorb lysis buffer andpurified using the Invisorb spin cell-RNA kit (INVITEK). Biotin-labeledcRNA was prepared following Affymetrix protocols and 15 μg cRNA werehybridized onto Affymetrix GeneChip set HG-U95.

[0159] Data Analysis

[0160] Raw data were analyzed using Affymetrix GeneChip softwareMicroarray Suite v4.0. The intensity of each probe set is calculated asdifference of the hybridization signal of perfect match oligonucleotidescompared to mismatch oligonucleotides averaged over the set of 16 to 20probe pairs corresponding to one transcript. The average difference of aprobe set is proportional to the abundance of a transcript. Total signalintensities of different arrays were scaled to the same value beforecomparison. Fold changes were calculated using the Affymetrix softwareby pairwise comparison of the intensities of corresponding probe pairsfrom experiment and baseline arrays. Using decision matrices describedby Affymetrix the software also generates absolute calls (transcript isabsent, marginal or present in an experiment) and difference calls(abundance of a transcript in one experiment compared to another:increase, marginal increase, no change, marginal decrease, decrease).Results were exported to Microsoft Excel (absolute call, differencecall, fold change) and filtered. All probe sets with absent calls or ano change call were discarded and the table sorted by the fold change.

[0161] Animal Studies

[0162] The in vivo experiments were conducted corresponding the GoodLaboratory Practice for Nonclinical Laboratory Studies (GLP Regulations)of the Food and Drug Administration and in accordance with the Germananimal protection law as legal basis.

[0163] Male Shoe:NMRI-nu/nu mice (Tierzucht Sch6nwalde GmbH) maintainedunder SPF conditions (Laminar air flow equipment, Scantainer, Scanbur)served as recipients for the human prostate carcinoma cells. Theanimals, aged 6-8 weeks and weighing 28-30 g, were inoculated 2×10⁶/0,03ml tumor cells into both, the left dorsolateral lobe of the prostaticgland (iprost; Orthotopic) or the tip of the Lobus lateralis sinister ofthe liver (ihep; Ectopic). For this purpose, the mice received a totalbody anaesthesia using a mixture of Ketanest (Parke-Davis GmbH) andRompun (Bayer Vital GmbH) 80:1 with dosages of 100 mg/kg and 5 mg/kg,respectively. Following the thorough sterilization of the ventral bodysurface an incision was carried out through the abdominal skin andperitoneal wall beginning near the border of the preputial gland andmeasuring about 1 cm. By means of a pair of tweezers and a cotton swabthe prostatic gland was visualized. The orthotopic cell challengesfollowed with the help of a magnifying glas and by usage of a 1 mlsyringe (Henke Sass Wolf GmbH) bearing 30G 0,30×13 microlance needles(Becton Dickinson). An administration was successful observing a markedbleb at the inoculation site. The wound was closed by suture material(PGA Resorba, Franz Hiltner GmbH) concerning the peritoneal wall andMichel clamps 11×2 mm (Heiland) for the abdominal skin. Wound spray(Hansaplast Spruhpflaster, Beiersdorf AG) covered the lesion. During thepostsurgical phase the animals were maintained in a warmed environmentuntil the complete waking up. The animals were randomised according tothe number of treatment groups consisting of 5-10 animals per groupeach. They were inspected successively inclusive of protocolling thefindings. Ssniff NM-Z, 10 mm, autoclavable (ssniff Spezialdiaten GmbH)is administered as fortified diet and drinking water is acidified byHCl, both ad libitum.

[0164] Evaluations

[0165] To receive the actual dosage level body weights were registeredon the treatment days. At the same time, it can be derived from bodyweight development to recognize influences of treatment modalities onthe whole organism.

[0166] Blood punctures were carried out on day 0 (Base line); 14; 28;and 35 (Sacrificing). Blood was drawn from the orbital vein of the shortterm anaesthesized animal (Diethylether, Otto Fischar GmbH). Evaluationparameters giving data to the compatibility and side effects of thetreatments are the following: Leukocyte numbers; Thrombocyte numbers;Enzymes. Further blood borne parameters were bilirubin; creatinine;protein; urea; uric acid.

[0167] All sacrificed animals were completely dissected andphotographically documented. Tumors (Prostatic gland) and metastases(Caudal, lumbar, renal lymph node metastases) were measured in twodimensions by means of a pair of callipers. The volume was calculatedaccording to V (mm³)=ab²/2 with b<a. In general, the cell numberperformed for therapy approaches causes a 100% tumor take concerning theprostatic gland. The weights of some organs (Liver; Spleen; Kidney) wereregistered in order to find out additional data concerning the knowledgeabout secondary side effects.

[0168] For histological analysis samples of tumor tissues, i.e. prostatetumor and lymph node metastases, were fixed in 5% formaldehyde andparaffin embedded. Routinely, the sections were HE stained, if necessaryspecific stainings were made (Azan, PAS).

[0169] To detect the human origin of tumor and metastatic cells adequatetissue samples were frozen in liquid nitrogen. When using PCR and Taqmananalysis with huHPRT specific amplicon we could detect 50 human cells inSmg tissue.

[0170] The therapeutic results were statistically verified by the u-testof Mann and Whitney.

EXAMPLE 2

[0171] Experimental Proof-Of-Concept on the Suitability of DownstreamDrug Targets

[0172] As outlined in the introductory part of this specification whichis incorporated herein by reference, targets linked downstream to asignalling pathway are valuable for the design or development of bothmedicaments and diagnostic agents. It is obvious that, if the particulartarget is linked to different other pathways or due to its positionwithin the signalling pathway is linked to a number of biologicalphenomena such as, e.g. metastasis and migration, growth translationapoptosis, cell cycle, DNA repair and the like as in the case of PI-3kinase, any compound addressing this target is likely to have a numberof side effects which may be detrimental to the system and undesiredfrom the medical point of view. Accordingly, targets that act furtherdownstream should be the first choice for therapeutic intervention.

[0173] The present inventors have found that under the control of the PI3-kinase pathway further possible drug targets apart from mTOR areinvolved, which are specific for controlling the phenomena of metastasisand migration and thus tumorigenesis. In the pharmaceutical industry ithas been found that rapamycin, sold under the trade name of Rapamune issuitable to inhibit metastasis and migration. This confirms thesuitability of the strategy to address downstream drug targets.

[0174] As may be taken from FIG. 2 rapamycin is suitable to reduce thevolume of lymphnode metastasis and is insofar comparable in its effectto the well known PI 3-kinase inhibitor LY294002. As depicted in FIG. 2A the tumor take model was used and treatment with Rapamune started onday 1. Both concentrations used, i.e. 0.4 mg/kg/dose-2 mg/kg/dose led toa tremendous decrease of the extent of lymphnode metastasis, expressedas measured volume of metastasis (mm³) compared to the negative controlwhich was phosphate buffered saline.

[0175] For histological analysis samples of tumor tissues, i.e. prostatetumor and lymph node metastases, were fixed in 5% formaldehyde andparaffin embedded. Routinely, the sections were HE stained, if necessaryspecific stainings were made (Azan, PAS).

[0176] The same results were basically also obtained in case of Rapamunetreatment of an established tumor model with the treatment starting onday 28.

[0177] Lymph node metastasis in an orthotopic PC-3 mouse model aftertreatment with rapamycin (Rapamune) was measured. In FIG. 2 (A) theresults of the (A), tumor take model are shown. Nude Shoe:NMRI—nu/numice (8 per group) were injected with 2×10⁶ PC3 cells in 0.03 mlintraprostatic and treatment was carried out using Rapamuneintraperitoneally daily for 28 days at doses of 2 mg/kg and 0.4 mg/kg.PBS served as a control.

[0178] For the treatment of established tumors (B), cells were allowedto grow ipros for 28 days and treatment was carried out orally usingRapamune on days 29 to 50 after implantation. Doses were chosen asoutline in A. Animals were sacrificed on day 29 and 51, respectively andtotal lymph node metastasis were determined

EXAMPLE 3

[0179] Identification of PKN Beta as Downstream Drug Target Within thePI 3-Kinase Pathway

[0180] The basic experimental approach is shown in FIG. 3. PC3 cellsgrown on Matrigel were either treated with DMSO or the PI 3-K inhibitorLY294002 and total RNA was isolated from each sample. DifferentialAffymetrix gene expression profiling was performed and expression wasconfirmned using real time RT-PCR Taqman assay. p110α was used as anon-differential standard. PC3 cells are PTEN −/− which means that thetumor suppressor PTEN is factually lacking in these cells so that the PI3-kinase pathway is permanently activated which leads to an increasedmetastatic activity or behaviour of the cells which is expressed bytheir growth pattern in the matrigel assay. Cells with invasive growthpotential exhibit enhanced growth on basement membrane such as matrigelmatrix. (Petersen, O. W., Ronnov-Jessen, L., Howlett, A. R. and Bissell,M. J. (1992) Interaction with basement membrane serves to rapidlydistinguish growth and differentiation pattern of normal and malignanthuman breast epithelial cells. Proc Natl Acad Sci USA, 89, 9064-9068.(Auch: Stemberger et al., 2002 Antisense & Nucleic acid drug development12:131-143)

[0181] In connection therewith it is to be noted that the PC3 cells weregrown on matrigel and taken this as a model system which is close to thein vivo environment the RNA isolated therefrom is assumed to be closerto the in situ situation or results than any preparation obtained fromcells grown in a non-matrigel environment such as a conventional cellculture plate.

EXAMPLE 4

[0182] Screening for Optimum Antisense Oligonucleotides Directed toProtein Kinase N Beta.

[0183] PC3 cells were transfected with different GeneBloc concentrationsas described and mRNA levels were determined 24 hrs post transfectionusing Taqman assays with 300 nM of PKNbeta specific forward and reverseprimer and 100 nM probe and 40 nM forward and reverse primer and 100 nMprobe for human β-actin. mRNA levels are standardized to internal actinlevels and amounts are shown relative to GBC (cells transfected with aGene Bloc Control).

[0184] The result thereof is shown in FIG. 4. From FIG. 4 asparticularly advantageous antisense oligonucleotides GeneBlocs 70210 and70211 were selected for further studies.

[0185] In connection with the GeneBloc as used herein in the variousexamples it is to be noted that they are all third generation antisenseoligonucleotide as specified herein which means, as also obvious fromtable 1, that the upper case letters represent the deoxyribonucleotideswhich were linked through a phosphorothioate rather than aphosphodiester linkage TABLE 1 Overview of the various GeneBlocs used,their alias, mismatches relative to the target nucleic acid and theirsequence and structured characteristics GeneBloc No Alias MM Sequence70669 PKNbeta:706L21 0 ggagguCCAGTTTCTgagagg 70670 PKNbeta:377L21 0uguuucACCTTCAGCuccaca 24536 PKNbeta:2021L23 0 aggacaaCACAAGCCAcgtagaa24537 PKNbeta:2665L23 0 gctctgaCACAAAGTCgaagtcc 24538 PKNbeta:2322L23 0gcagtcaAACACCTCTtcctctg 70210 PKNbeta:1034L21 0 caacacGGTTGTCCAccttta70211 PKNbeta:1784L21 0 tcagtgCTTTGATGGcgtagt 70671 PKNbeta:183L21 0cuucucGCAGTACAGgcucuc 70676 PKNbeta:1034L21 4 caagacGCTTGTGCAcgttta70677 PKNbeta:1784L21 4 tcagagCTTAGTTGGcgttgt

[0186] The various GeneBlocs correspond to the following SEQ. ID. Nos:70669: SEQ. ID. No. 3 70670: SEQ. ID. No. 4 24536: SEQ. ID. No. 5 24537:SEQ. ID. No. 6 24538: SEQ. ID. No. 7 70210: SEQ. ID. No. 8 70211: SEQ.ID. No. 9 70671: SEQ. ID. No. 10 70676: SEQ. ID. No. 11 70677: SEQ. ID.No. 12

[0187] In addition it is to be noted that any of the “t” above areactually “u” given the fact that the above antisense oligonucleotidesare GeneBlocs, i.e. third generation antisense oligonucleotides.

EXAMPLE 5

[0188] Selective Knock Down of Protein Kinase N Beta

[0189] In order to prove that protein kinase N beta is a suitabledownstream drug target of the PI 3-kinase pathway the two particularlyadvantageous GeneBlocs as obtained from example 4 were used in amatrigel based growth experiment. The matrigel growth experiment istaken as a surrogate model which shows the metastasis and migrationbehaviour of the respective cell. A more confluent growth of the cellsis taken as an indication that their metastasis and migration behaviouris increased which allows the cells to spread over the three-dimensionalstructure provided by the matrigel.

[0190] PC3 cells were transfected and seeded on matrigel as describedand growth was monitored. siRNA was isolated from an aliquot of thecells seeded on matrigel and analysed using Taqman assay (left panel).PKNbeta specific mRNA was standardized to endogenous p110α mRNA levels.A PTEN specific GeneBloc is used as a negative control in the PTEN^(−/−)PC-3 cells and a p110β specific GeneBloc is used as a positive controlfor growth in extracellular matrix. Specific growth inhibition is shownby comparing growth of cells treated with PKN beta specific GeneBloc70210 or 70211 versus their corresponding mismatched oligonucleotides70676 and 70677, respectively.

[0191] The respective results are also illustrated in FIG. 5. From thisit may be taken that the gene block 70211 and 70210 may be suitablecompounds for the manufacture of a medicament or diagnostic agent forthe treatment of diseases and diseased conditions as described herein.

EXAMPLE 6

[0192] RNA Interference by Transient Expression of siRNA in HeLaB Cells

[0193] This experiment is an example of the successful design of siRNAwhich allows that specifically the downstream drug target protein kinaseN beta is addressed. As illustrated in FIG. 6 (A) siRNA molecules weregenerated by promoter (U6+2) driven expression of target specificsequences (template derived from gene of interest containing a 21-mersense and reverse complementary sequences linked by 12-mer poly Astretch. Upon transcription RNAs are likely to form double-strandedsiRNA molecules.

[0194] The various constructs such as p110beta and PTEN were used aspositive and negative control, respectively in the same vector constructas the siRNA designed against the mRNA sequence of PKNbeta. Therespective design is shown in FIG. 6 (B) were the template sequences oftargeted genes for siRNA expression were introduced into expressionvectors carrying the U6+2 promoter cassette.

[0195] The constructs were transiently expressed by transfection intoHeLaB cells for RNAi interference experiments. Cells were harvested 48hour after transfected and subsequently seeded (80000 cells per well) onmatrigel. The effect of RNA interference on the expression ofcorresponding genes was analyzed by assaying transfected cells forgrowth/proliferation on matrigel. Expression of siRNA targeted to PTENhad no affect on HeLaB cell growth on matrigel (right panel), whereasexpression of siRNA specific to p11 Obeta and PKNbeta severely disturbedthe behaviour of HeLaB growth on matrigel (middle and right panels).

[0196] In view of this, the particular siRNA sequence proves to be anefficient means for the treatment of the disease and disease conditionsas disclosed herein.

EXAMPLE 7

[0197] Detection of Protein Kinase N Beta in Human Prostate Tumor

[0198] In order to give further evidence that protein kinase N beta is asuitable target in the treatment of prostate tumor, respective humanprostate tissue was subjected to in situ hybridisation.

[0199] For the in situ hybridisation both sense and antisense strandswere prepared from nucleotide positions 1672 to 2667 from sequence NM013355 in pCR4 Topo vector, whereupon T7 and T3 polymerase was used foramplification purposes. The human prostate tumor cells (PC-3) were grownin mice. After dissection, the tissue was frozen at −20° C. inisopentane solution, slices cut at −15° C. and stored at −80° C. Priorto hybridisation slices were fixed in paraformaldehyde. Human tumorspecimen were fixed in paraformaldehyde and paraffine-embedded. Tumorspecimens were treated with proteinase K and acetylated. Nucleic acidprobes were double-labelled with ³⁵S-ATP and ³⁵S-UTP and incubated withtissues at 58° C. in a hybridisation buffer (0.4 M NaCl, 50% formamide,1× Denhardt's, 10 mM Tris, 1 mM_EDTA, 10% dextran sulfate, 10 μg/ml ofeach tRNA and salmon sperm DNA, 10 mM DTT) containing 50% formamide.

[0200] The result of the in situ hybridisation is depicted in FIG. 7.Using protein kinase N beta antisense probe for in situ hybridisation ofprostate tumor the glands are intensively stained (FIG. 7A). In contrastto this, healthy prostate tissue is less stained and provides for abackground signal only, again using the antisense probe (FIG. 7C). Incontrast to this, the use of the sense probe in connection with bothtissues, did not provide any signal.

EXAMPLE 8

[0201] In Vivo Reduction of Primary Tumor and Lymph Node Metastases bysiRNA

[0202] This example is related to target gene validation in vivo usingan orthotopic prostate tumor model in which it could be shown that byusing siRNA directed to protein kinase N beta a reduction of bothprimary tumor and lymph node metastases could be realised. The resultsare depicted in FIGS. 8A to 8C.

[0203] In the diagram of FIG. 8A the volume of primary tumors,determined as described in example 1, in an orthotopic prostate tumormodel could be significantly reduced using any of the following twosiRNA constructs: 5′actgagcaagaggctttggag or 5′aaattccagtggttcattcca.

[0204] As negative control siRNA against p110-α subunit was used and aspositive control siRNA against p110-β subunit. The positive control thusaddresses the upstream regulator of protein kinase N beta PTEN.

[0205] A further set of two independent siRNA molecules was used fordegrading the MRNA encoding for protein kinase N beta in lymph nodemetastases. Lymph node metastases are secondary tumors found in thefollowing lymph nodes: Caudal, Lumbar, Renal and mediastinal lymph nodeswhereby caudal lymph nodes are closest to the prostate and mediastinallymph nodes are most distanct to the implantation tumor. As in the caseof the primary tumor, the siRNA constructs were obviously successfullyreducing the mRNA coding for protein kinase N beta and thus reducing thetumor volume (FIG. 8B). Positive and negative controls were as discussedin connection with the reduction of primary tumor.

[0206] In both cases, i.e. for primary tumor and lymph node metastases,the human prostate tumor cells were genetically engineered to expressthe respective siRNA molecules from a polymerase III U6 promoter.

[0207] Apart from these results, a clear phenotypic analysis as depictedin FIG. 8C1 and FIG. 8C2 shows that upon activation of the transcriptionof the siRNA construct in the human prostate tumor cells, lymph nodemetastases could be significantly reduced and the swollen lymph nodedepicted in FIG. 8C1 is not present in the tissue treated with siRNA asdepicted in FIG. 8C2.

EXAMPLE 9

[0208] Functional Characterisation of the Protein Kinase N Beta

[0209] This example is related to the functional characterisation ofprotein kinase N beta and more particularly to the impact ofderivatisation, i.e. truncation or mutation of fluctional amino acidresidues, of protein kinase N beta on its kinase activity and on theregulation of its kinase activity by phosphorylation.

[0210] The following protein kinase N beta derivatives were generated asalso at least partially depicted schematically in FIG. 9C with the aminoacid residues referring to the wild type sequence as disclosed herein:

[0211] a) kinase domain comprising amino acids 535-889;

[0212] b) AN comprising amino acids 288-889;

[0213] c) kinase domain having a mutation at position 588 from lysine toarginine;

[0214] d) kinase domain having a mutation at position 588 from lysine toglutamic acid;

[0215] derivatives of the kinase domain having mutations at thephosphorylation site (AGC activation loop consensus) with the threonineresidue at amino acid position 718 being either changed to alanine(TA718) or to aspartic acid or glutamic acid (TD718 or TE718); and

[0216] full length wildtype PKNbeta molecule (889 amino acids).

[0217] The respective fragments were transiently epressed in HeLa cells.Their relative expression was determined by Western-blot analysis ofHela cell extracts using an anti-PKNbeta antibody.

[0218] The polyclonal anti-PKNbeta antiserum was generated afteroverexpressing the C-terminal amino acids (609-889)of PKNbeta in E.coli. The respective protein fragment was gel-purified from inclusionbodies, recovered and concentrated according to standard procedures.

[0219] Protein kinase N beta has homologies to AGC-type kinase moleculesin its catalytic domain at the C-terminus. The family of kinases ischaracterised by a conserved threonine residue in the activation loop ofthe catalytic domain that needs to be phosphorylated for enzymaticactivity. Due the high conservation of this threonine and thesurrounding amino acid context in the activation loop, anti-phosphoantibodies against this site are available from commercial sources. Therespective antibodies are referred to as anit-P*-PRK in FIG. 9 and asanti-P*-AGC kinase in FIG. 10.

[0220] MPB is myelin basic protein which is a standard in vitrophosphorylation substrate.

[0221] The following results were obtained: Protein kinase N betaderivative Activity Full length wt ++* Kinase domain comprising aminoacids 535-889 +* ΔN comprising amino acids 288-889 −* Kinase domainhaving a mutation at position 588 from lysine to −* arginine (KR 588)Kinase domain having a mutation at position 588 from lysine to −*glutamic acid (KR 588) TA718 −* TD718 or TE718 −*!

[0222] The results are depicted in FIG. 9.

[0223]FIG. 9A shows a gel analysis of different protein kinase N betaderivatives and their activities using MPB as a standard phosphorylationsubstrate upon transient overexpression in HeLa cells. As may be takenfrom FIG. 9A apart from the full-length protein kinase N beta only thekinase domain in its otherwise wildtype form is active inphosphorylating MPB.

[0224] Using the same protein kinase N beta derivatives it can beobserved that except the derivative comprising kinase domain having themutation T/A at position 718, all other derivatives displayed were alsophosphorylated regardless of their further intrinsic activities.

[0225] The data indicates that the presence of a functional kinasedomain and phosphorylation at position 718 are pre-requisites forPKNbeta kinase activity. However, as can be concluded from the inabilityof the AN version to act as a kinase, they are not sufficient. The dataalso indicates that PKNbeta does not autophosphorylate at amino acid718, but instead, requires phosphorylation by another kinase molecule,since the kinase defective KR588 mutant protein retains phosphorylationat position 718.

EXAMPLE 10

[0226] Characterization of Full Length PKNbeta

[0227] In order to analyse mutations of functional amino acid residuesof protein kinase N beta in the context of the full length molecule, thefollowing experiments were carried out as shown in FIGS. 10 and 11:

[0228] For measuring the kinase activity of PKNbeta in vitro,recombinant HA- or Myc-tagged PKNbeta derivatives were transientlyexpressed in HeLa or COS-7 cells. The smaller kinase domain derivativeserved as a control. The cell extracts containing the recombinantversions of protein kinase N beta were probed in parallel withanti-protein kinase N-beta antibody (FIG. 1OA) as described in example 9to demonstrate comparable expression levels, and an anti-phospho AGCsite antibody (also referred to as anti-P*-AGC-kinase) (FIG. 10B) toshow the different degree of phosphorylation of the protein kinase Nbeta derivatives in vivo.

EXAMPLE 11

[0229] Phosphorylation Requirements for the Activity of Full LengthProtein Kinase N Beta and Development of a Non-RadioactiveIn-Vitro-Kinase Assay—Suitability of Protein Kinase N Beta for HTSAssays

[0230] The PKNbeta-derived molecules were immune-precipitated from thecell extracts shown in FIG. 10 by using anti-tag antibodies. The immuneprecipitates were washed as described (Klippel et al., 1996) and dividedin two halfs. One half was incubated with 5 μg MBP (UBI) as aphosphorylation substrate, 4 mM MgCl₂ and gamma ³²P-ATP in a bufferedsolution for 10 min at room temperature. In addition, phosphataseinhibitors and inhibitors against unspecifically acting kinases wereadded as in Klippel et al., 1998. Incorporation of radioactive phosphatewas detected by autoradiography after separating the reaction productsby 16% SDS-PAGE (FIG. 11B).

[0231] The second half of the immune precipitates was incubated with 1μg GST-GSK3 fusion protein (Cell Signaling Technology) as aphosphorylation substrate in the presence of 200 μM rATP. The reactionmixture was subsequently analyzed by 8-16% gradient SDS-PAGE and Westernblotting using the anti-phospho GSK3alpha antibody (Cell SignalingTechnology) (FIG. 11A). The filter was then stripped and re-probed withanti-PKNbeta antiserum to confirm the presence of comparable amounts ofPKNbeta proteins in the respective immune precipitates (FIG. 11C).

[0232] The specificity of the in vitro phosphorylation reactions wascontrolled by analyzing kinase defective variants (e.g. containingmutations in the ATP binding site, see above) in parallel to activeprotein.

[0233] The lack of signal in case of the TA mutation variant at aminoacid 718 of protein kinase N beta otherwise being the full lengthwildtype protein kinase N beta indicates that this amino acid residue isindeed the position of phosphorylation detected by the antibody (FIG.10B). The fact that the kinase deficient variants (KE or KR mutation asshown in FIG. 10 and FIG. 9, respectively) are phosphorylated at thissite indicates that threonine 718 is not a substrate forautophosphorylation. Rather another kinase in the cells must beresponsible for phosphorylation of this site; whereby PDK1 is a possiblecandidate.

[0234] Also, from this experiment in combination with the one of example9 it is revealed that phosphorylation of protein kinase N beta atposition 718 is pre-requisite for protein kinase N beta activity; allmutations tested at this site prevented phosphorylation and resulted inan inactive kinase molecule. Insofar a particularly preferred proteinkinase N beta which can be used in connection with any aspects of theinvention as disclosed herein is a protein kinase N beta beingphosphorylated at position 718 or a derivative thereof, including thederivative which comprises the kinase domain only as described herein.The data further indicates that also full length PKNbeta does notautophosphorylate at amino acid 718, but instead, requiresphosphorylation by another kinase molecule, since the kinase defectiveKE588 mutant protein retains phosphorylation at position 718.

[0235] As may be taken from FIG. 11, assaying the activity of proteinkinase N beta can be adapted into a format which allows the screening ofprotein kinase N beta inhibitors into a high throughput system.

[0236] In a first step, the suitability of a non-radioactive screeningformate was determined, whereby the various protein kinase N betaderivatives as already discussed in connection with example 10, wereused for phosphorylating a suitable substrate. Such substrate may, forexample, be MBP or a GSK3 peptide which is typically immobilised on asuitable carrier such as agarose- or sepharose beads or on plasticsurfaces. In the present case and as depicted in FIG. 11A, the substrateis a GSK3-derived peptide fused to paramyosin. The first row indicatesthat all of the various assays using different protein kinase N betaderivatives actually contained said derivatives. Only the full lengthwildtype protein kinase N beta or the kinase domain as defined inexample 9 were suitable to phosphorylate the substrate. Thephosphorylated substrate in the present case was detected byanti-phospho GSK3 alpha antibody (mentioned above).

[0237] To make sure that the non-radioactive approach as depicted inFIG. 11A is sensitive enough the radioactive approach was carried out inparallel with half of the immune precipitates using the MBP as aphosphorylation substrate. The efficacy of the kinase activity can betaken from the amount of the generated phosphorylated substrate asindicated by autoradiography upon [³²P] incorporation. As can be seenfrom FIGS. 11A and 11B, the full length wildtype protein kinase N betaas well as the kinase domain show activity, whereas no (FIG. 11A) orbackground activity of unspecific kinases (FIG. 11B) were detected withthe full length KE and full length TA mutatant proteinss, respectively.

[0238] To summarise, the use of both full length wildtype protein kinaseN beta as well as the kinase domain as disclosed herein, are suitabletargets or means for the design of a screening procedure in HTS format.The respective steps would accordingly comprise

[0239] a) generating purified recombinant protein kinase N beta proteinby expression in a non-bacterial expression system such as insect cellsystem (example for different kinase in Klippel et al., 1997) in view ofthe fact that the protein needs to be phosphorylated for exhibitingkinase activity, which cannot easily be accomplished by expression inbacterial systems;

[0240] b) immobilisation of GSK3-derived substrate or similar substrate,and incubating the substrate with purified protein kinase N beta in thepresence of rATP, MgCl₂ and inhibitors in a buffered solution;

[0241] c) detecting phosphorylation of the substrate by an appropriatedetection means such as an antibody like the anti-phopho-GSK3 antibodyoptionally upon after serial washes and further optionally subsequentlydeveloping in Delfia or Lance assay systems (Perkin Elmer), whereby thephosphorylation site is bound by a Europium-labelled antibody. Theamount of bound Europium is then quantitated by time-resolvedfluorescence analysis.

EXAMPLE 12

[0242] Determination of the Expression Level of Endogenous PKN-Beta

[0243] In this example experimental evidence is given that PKN-beta isexpressed in a P13-kinase dependent manner. The PI 3-kinase-dependentexpression of PKNbeta RNA shown in FIG. 3 is here further confirmed onprotein level.

[0244] PC-3 cells were cultivated as described in example 1 herein. SaidPC-3 cells are PTEN −/−. HeLa cells were obtained from the American TypeCulture Collection (ATCC) and grown as described in Sternberger et al.(2002). Transfections were carried out in 10-cm plates (at 30% to 50%confluency) using Fugene 6 (Roche, Nutley, N.J.) according to themanufacturer's instructions. Cultivated cells were trypsinated andharvested following stopping the trypsin effect by medium.

[0245] Both cell types, i.e. PC-3 cells and HeLa cells were treated forthe indicated times with 10 gM LY294002 or DMSO, whereby DMSO was usedas the solvent for LY294002 and, because of this, as negative control.

[0246] The resulting extracts were fractionated by SDS-PAGE andsubsequently analysed by Western-blotting. The levels of the indicatedproteins such as p110, which served as a loading control, endogenousPKN-beta and phosphorylated Akt were detected using the respectiveantibodies. Phosphorylated Akt (P*-Akt) serves as a control for theefficacy of the LY294002-mediated treatment.

[0247] The results are depicted in FIG. 12.

[0248] In PC-3 cells inhibition of PI-3-kinase caused a visiblereduction of endogenous PKNbeta expression after 24 h, the proteinlevels were further reduced after 48 h treatment. In HeLa cells, whichexpress higher amounts of PKNbeta protein, this effect is less dramatic,but reduced amounts can be detected after 48 h treatment with LY294002.

[0249] From this it can be concluded, that P13-kinase controls theexpression of PKN-beta.

EXAMPLE 13

[0250] PKN-Beta Activity Requires P13-Kinase

[0251] Recombinant wildtype PKN-beta or derivatives of PKN-beta (asdescribed in FIGS. 10-11) were transiently expressed in HeLa cells. Saidderivatives were PKN-beta derivative TA and derivative KE as describedin example 10 herein. The PKN-beta was modified in each case by amyc-tag as described above which allowed the precipitation of PKN-betaand its derivatives using an anti-Myc antibody.

[0252] For the assessment of the activity of PKN-beta an in vitro kinaseactivity using the immune precipitates was carried out as describedabove. Half of the precipitates were subjected to the in vitro kinasereaction, the second half was analyzed by Western-blotting usinganti-phospho-PRK antibodies. The filter was stripped and reprobed usingthe anti-PKN-beta antiserum. Phospho-p70 S6 Kinase levels were analyzedfrom aliquots of cell lysates, that were withdrawn earlier, to confirmthe efficacy of the LY294002 treatment even after only 3 h treatment.The anti-phospho p70 antibody was obtained from Cell signaling.

[0253] As can be seen from FIG. 13 LY294002 treatment leads to stronginhibition of the kinase activity of PKNbeta, measured here again byphosphorylation of MBP. This effect was visible after only 3 h oftreatment, at 24 h PKNbeta activity was almost completely inhibited. Thephosphorylation of PKNbeta at position 718 was also compromised afterinhibition of PI 3-kinase by LY294002, however, this effect was lesspronounced than the effect on the kinase activity.

[0254] The TA derivative of PKN-beta serves as inactive control as shownabove, and as control for the specificity of the anti-phospho PRKantibody for phospho-threonine at position 718 (P*-PK)

[0255] PKN-beta derivative KE serves as kinase-deficient control asdescribed above. Its phosphorylation status appeared also to some extentaffected by LY294002 treatment. This indicates that the kinase, which isresponsible for phosphorylating PKNbeta at position 718, does so in a PI3-kinase-dependent fashion.

[0256] Most importantly, this experiment shows that PKNbeta is not onlyregulated by PI 3-kinase via its expression level (see FIGS. 3 and 12),it is also regulated at its activation level. These findings indicatethat PKNbeta represents a “perfect” downstream target for interferencewith a hyperactive PI 3-kinase pathway for therapeutic intervention,since it is equisitely dependent on PI 3-kinase being regulated by it atvarious levels. This allows the generation of compounds which exhibit adistinct effect on both the protein PKN-beta and the nucleic acid codingtherefor. Even more important, this kind of activity modulation ofPKN-beta at the translation rather than transcription level, i.e. at thelevel of the expressed protein, seems to be more prominent and longerlasting than the impact at the transcription level.

[0257] The further screening method according to the present inventionis based on this particular insight and uses preferably the radioactiveor nonradioactive in vitro kinase assay as read-out.

EXAMPLE 14

[0258] Localization Signals of PKN-Beta

[0259] In this experiment the localization of various PKN-betaderivatives was compared to the localization of wildtype PKN-beta. FIG.14 shows pictures, whereby the cellular distribution of PKNbeta andderivatives thereof such as PKN beta wildtype (FIG. 14A), PKN betaderivative TA (FIG. 14B), PKN beta derivative KE (FIG. 14C) and PKN betadeltaN (FIG. 14D) was investigated by confocal fluorescence microscopy.HA-tagged recombinant derivatives of PKNbeta were transiently expressedin HeLa cells for 48 h. After fixing and perneabilization, expression ofthe recombinant proteins was detected by using an anti-HA antibodyfollowed by an FITC-conjugated anti-mouse antibody. The cells werecounterstained by labelling the cytoskeletal actin withrhodamin-phalloidin.

[0260] The results are depicted in FIGS. 14A to 14D, whereby therespective picture on each left side of the duplex of the pictures isrelated to a picture of cells upon FITC-specific excitation and theright picture illustrates the same cells upon excitation using awavelength specific for Rhodamin-phalloidin. The FITC-staining indicatescells transfected with the respective recombinant protein. TheRhodamin-phalloidin staining shows transfected and unstransfected cells.

[0261] As may be taken from FIG. 14A, wildtype PKN-beta localizespredominantly to the nucleus of the cells. The phosphorylation sitemutant of PKN-beta TA and the KE mutant, both are kinase-deficient, areno longer concentrated within the nucleus compared to wildtype PKN-beta,but rather spread over the entire cell. Finally, as depicted in FIG. 14Dthe PKN-beta derivative ΔN, which lacks the N-terminal third of themolecule and is also kinase-defective (see FIG. 9), is essentiallyexcluded from the nucleus.

[0262] These data indicate that proper nuclear localization of PKNbetato the nucleus is dependent on its ability to act as an active kinasemolecule and involves the presence of its N-terminal domain. Thisimplicates that PI-3 kinase might regulate also its cellularlocalization.

[0263] The features of the present invention disclosed in thespecification, the sequence listing, the claims and/or the drawings mayboth separately and in any combination thereof be material for realizingthe invention in various forms thereof.

1 12 1 889 PRT Homo sapiens MISC_FEATURE (1)..(889) protein kinase Nbeta (PKN beta) 1 Met Glu Glu Gly Ala Pro Arg Gln Pro Gly Pro Ser GlnTrp Pro Pro 1 5 10 15 Glu Asp Glu Lys Glu Val Ile Arg Arg Ala Ile GlnLys Glu Leu Lys 20 25 30 Ile Lys Glu Gly Val Glu Asn Leu Arg Arg Val AlaThr Asp Arg Arg 35 40 45 His Leu Gly His Val Gln Gln Leu Leu Arg Ser SerAsn Arg Arg Leu 50 55 60 Glu Gln Leu His Gly Glu Leu Arg Glu Leu His AlaArg Ile Leu Leu 65 70 75 80 Pro Gly Pro Gly Pro Gly Pro Ala Glu Pro ValAla Ser Gly Pro Arg 85 90 95 Pro Trp Ala Glu Gln Leu Arg Ala Arg His LeuGlu Ala Leu Arg Arg 100 105 110 Gln Leu His Val Glu Leu Lys Val Lys GlnGly Ala Glu Asn Met Thr 115 120 125 His Thr Cys Ala Ser Gly Thr Pro LysGlu Arg Lys Leu Leu Ala Ala 130 135 140 Ala Gln Gln Met Leu Arg Asp SerGln Leu Lys Val Ala Leu Leu Arg 145 150 155 160 Met Lys Ile Ser Ser LeuGlu Ala Ser Gly Ser Pro Glu Pro Gly Pro 165 170 175 Glu Leu Leu Ala GluGlu Leu Gln His Arg Leu His Val Glu Ala Ala 180 185 190 Val Ala Glu GlyAla Lys Asn Val Val Lys Leu Leu Ser Ser Arg Arg 195 200 205 Thr Gln AspArg Lys Ala Leu Ala Glu Ala Gln Ala Gln Leu Gln Glu 210 215 220 Ser SerGln Lys Leu Asp Leu Leu Arg Leu Ala Leu Glu Gln Leu Leu 225 230 235 240Glu Gln Leu Pro Pro Ala His Pro Leu Arg Ser Arg Val Thr Arg Glu 245 250255 Leu Arg Ala Ala Val Pro Gly Tyr Pro Gln Pro Ser Gly Thr Pro Val 260265 270 Lys Pro Thr Ala Leu Thr Gly Thr Leu Gln Val Arg Leu Leu Gly Cys275 280 285 Glu Gln Leu Leu Thr Ala Val Pro Gly Arg Ser Pro Ala Ala AlaLeu 290 295 300 Ala Ser Ser Pro Ser Glu Gly Trp Leu Arg Thr Lys Ala LysHis Gln 305 310 315 320 Arg Gly Arg Gly Glu Leu Ala Ser Glu Val Leu AlaVal Leu Lys Val 325 330 335 Asp Asn Arg Val Val Gly Gln Thr Gly Trp GlyGln Val Ala Glu Gln 340 345 350 Ser Trp Asp Gln Thr Phe Val Ile Pro LeuGlu Arg Ala Arg Glu Leu 355 360 365 Glu Ile Gly Val His Trp Arg Asp TrpArg Gln Leu Cys Gly Val Ala 370 375 380 Phe Leu Arg Leu Glu Asp Phe LeuAsp Asn Ala Cys His Gln Leu Ser 385 390 395 400 Leu Ser Leu Val Pro GlnGly Leu Leu Phe Ala Gln Val Thr Phe Cys 405 410 415 Asp Pro Val Ile GluArg Arg Pro Arg Leu Gln Arg Gln Glu Arg Ile 420 425 430 Phe Ser Lys ArgArg Gly Gln Asp Phe Leu Arg Arg Ser Gln Met Asn 435 440 445 Leu Gly MetAla Ala Trp Gly Arg Leu Val Met Asn Leu Leu Pro Pro 450 455 460 Cys SerSer Pro Ser Thr Ile Ser Pro Pro Lys Gly Cys Pro Arg Thr 465 470 475 480Pro Thr Thr Leu Arg Glu Ala Ser Asp Pro Ala Thr Pro Ser Asn Phe 485 490495 Leu Pro Lys Lys Thr Pro Leu Gly Glu Glu Met Thr Pro Pro Pro Lys 500505 510 Pro Pro Arg Leu Tyr Leu Pro Gln Glu Pro Thr Ser Glu Glu Thr Pro515 520 525 Arg Thr Lys Arg Pro His Met Glu Pro Arg Thr Arg Arg Gly ProSer 530 535 540 Pro Pro Ala Ser Pro Thr Arg Lys Pro Pro Arg Leu Gln AspPhe Arg 545 550 555 560 Cys Leu Ala Val Leu Gly Arg Gly His Phe Gly LysVal Leu Leu Val 565 570 575 Gln Phe Lys Gly Thr Gly Lys Tyr Tyr Ala IleLys Ala Leu Lys Lys 580 585 590 Gln Glu Val Leu Ser Arg Asp Glu Ile GluSer Leu Tyr Cys Glu Lys 595 600 605 Arg Ile Leu Glu Ala Val Gly Cys ThrGly His Pro Phe Leu Leu Ser 610 615 620 Leu Leu Val Cys Phe Gln Thr SerSer His Ala Arg Phe Val Thr Glu 625 630 635 640 Phe Val Pro Gly Gly AspLeu Met Met Gln Ile His Glu Asp Val Phe 645 650 655 Pro Glu Pro Gln AlaArg Phe Tyr Val Ala Cys Val Val Leu Gly Leu 660 665 670 Gln Phe Leu HisGlu Lys Lys Ile Ile Tyr Arg Asp Leu Lys Leu Asp 675 680 685 Asn Leu LeuLeu Asp Ala Gln Gly Phe Leu Lys Ile Ala Asp Phe Gly 690 695 700 Leu CysLys Glu Gly Ile Gly Phe Gly Asp Arg Thr Ser Thr Phe Cys 705 710 715 720Gly Thr Pro Glu Phe Leu Ala Pro Glu Val Leu Thr Gln Glu Ala Tyr 725 730735 Thr Gln Ala Val Asp Trp Trp Ala Leu Gly Val Leu Leu Tyr Glu Met 740745 750 Leu Val Gly Glu Cys Pro Phe Pro Gly Asp Thr Glu Glu Glu Val Phe755 760 765 Asp Cys Ile Val Asn Met Asp Ala Pro Tyr Pro Gly Phe Leu SerVal 770 775 780 Gln Gly Leu Glu Phe Ile Gln Lys Leu Leu Gln Lys Cys ProGlu Lys 785 790 795 800 Arg Leu Gly Ala Gly Glu Gln Asp Ala Glu Glu IleLys Val Gln Pro 805 810 815 Phe Phe Arg Thr Thr Asn Trp Gln Ala Leu LeuAla Arg Thr Ile Gln 820 825 830 Pro Pro Phe Val Pro Thr Leu Cys Gly ProAla Asp Leu Arg Tyr Phe 835 840 845 Glu Gly Glu Phe Thr Gly Leu Pro ProAla Leu Thr Pro Pro Ala Pro 850 855 860 His Ser Leu Leu Thr Ala Arg GlnGln Ala Ala Phe Arg Asp Phe Asp 865 870 875 880 Phe Val Ser Glu Arg PheLeu Glu Pro 885 2 2670 DNA Homo sapiens mRNA (1)..(2670) mRNA / cDNA ofPKN beta 2 atggaggagg gggcgccgcg gcagcctggg ccgagccagt ggcccccagaggatgagaag 60 gaggtgatcc gccgggccat ccagaaagag ctgaagatca aggagggggtggagaacctg 120 cggcgcgtgg ccacagaccg ccgccacttg ggccatgtgc agcagctgctgcggtcctcc 180 aaccgccgcc tggagcagct gcatggcgag ctgcgggagc tgcacgcccgaatcctgctg 240 cccggccctg ggcctggccc agctgagcct gtggcctcag gaccccggccgtgggcagag 300 cagctcaggg ctcggcacct agaggctctc cggaggcagc tgcatgtggagctgaaggtg 360 aaacaggggg ctgagaacat gacccacacg tgcgccagtg gcacccccaaggagaggaag 420 ctccttgcag ctgcccagca gatgctgcgg gacagccagc tgaaggtggccctgctgcgg 480 atgaagatca gcagcctgga ggccagtggg tccccggagc cagggcctgagctactggcg 540 gaggagctac agcatcgact gcacgttgag gcagcggtgg ctgagggcgccaagaacgtg 600 gtgaaactgc ttagtagccg gagaacacag gaccgcaagg cactggctgaggcccaggcc 660 cagctacagg agtcctctca gaaactggac ctcctgcgcc tggccttggagcagctgctg 720 gagcaactgc ctcctgccca ccctttgcgc agcagagtga cccgagagttgcgggctgcg 780 gtgcctggat acccccagcc ttcagggaca cctgtgaagc ccaccgccctaacagggaca 840 ctgcaggtcc gcctcctggg ctgtgaacag ttgctgacag ccgtgcctgggcgctcccca 900 gcggccgcac tggccagcag cccctccgag ggctggcttc ggaccaaggccaagcaccag 960 cgtggccgag gcgagcttgc cagtgaggtg ctggctgtgc taaaggtggacaaccgtgtt 1020 gtggggcaga cgggctgggg gcaggtggcc gaacagtcct gggaccagacctttgtcatc 1080 ccactggagc gagcccgtga gctggagatt ggggtacact ggcgggactggcggcagcta 1140 tgtggcgtgg ccttcctgag acttgaagac ttcctggaca atgcctgtcaccaactgtcc 1200 ctcagcctgg taccgcaggg actgcttttt gcccaggtga ccttctgcgatcctgtcatt 1260 gagaggcggc cccggctgca gaggcaggaa cgcatcttct ctaaacgcagaggccaggac 1320 ttcctgaggc gttcgcagat gaacctcggc atggcggcct gggggcgcctcgtcatgaac 1380 ctgctgcccc cctgcagctc cccgagcaca atcagccccc ctaaaggatgccctcggacc 1440 ccaacaacac tgcgagaggc ctctgaccct gccactccca gtaatttcctgcccaagaag 1500 acccccttgg gtgaagagat gacaccccca cccaagcccc cacgcctctacctcccccag 1560 gagccaacat ccgaggagac tccgcgcacc aaacgtcccc atatggagcctaggactcga 1620 cgtgggccat ctccaccagc ctcccccacc aggaaacccc ctcggcttcaggacttccgc 1680 tgcttagctg tgctgggccg gggacacttt gggaaggtcc tcctggtccagttcaagggg 1740 acagggaaat actacgccat caaagcactg aagaagcagg aggtgctcagccgggacgag 1800 atagagagcc tgtactgcga gaagcggatc ctggaggctg tgggctgcacagggcaccct 1860 ttcctgctct ccctccttgt ctgcttccag acctccagcc atgcccgctttgtgactgag 1920 tttgtgcctg gtggtgacct catgatgcag atccacgagg atgtcttccccgagccccag 1980 gcccgcttct acgtggcttg tgttgtcctg gggctgcagt tcttacacgagaagaagatc 2040 atttacaggg acctgaagtt ggataacctt ctgctggatg cccagggattcctgaagatc 2100 gcagactttg gactctgcaa ggaagggatc ggcttcgggg accggactagcaccttctgt 2160 ggcaccccgg agttcctggc tcccgaggtg ctgacccagg aggcatacacacaggccgtc 2220 gactggtggg cgctgggtgt gctgctctac gagatgctgg tgggtgagtgcccgttccca 2280 ggggacacag aggaagaggt gtttgactgc atcgtcaaca tggacgccccctaccccggc 2340 tttctgtcgg tgcaagggct tgagttcatt cagaagctcc tccagaagtgcccggagaag 2400 cgcctcgggg caggtgagca ggatgccgag gagatcaagg tccagccattcttcaggacc 2460 accaactggc aagccctgct cgcccgcacc atccagcccc ccttcgtgcctaccctgtgt 2520 ggccctgcgg acctgcgcta ctttgagggc gagttcacag ggctgccgcctgccctgacc 2580 ccacctgcac cccacagcct cctcactgcc cgccaacagg ccgccttccgggacttcgac 2640 tttgtgtcag agcgattcct ggaaccctga 2670 3 21 DNAArtificial Sequence antisense oligonucleotide 3 ggagguccag tttctgagag g21 4 21 DNA Artificial Sequence antisense oligonucleotide 4 uguuucaccttcagcuccac a 21 5 23 DNA Artificial Sequence antisense oligonucleotide 5aggacaacac aagccacgua gaa 23 6 23 DNA Artificial Sequence antisenseoligonucleotide 6 gcucugacac aaagtcgaag ucc 23 7 23 DNA ArtificialSequence antisense oligonucleotide 7 gcagucaaac acctctuccu cug 23 8 21DNA Artificial Sequence antisense oligonucleotide 8 caacacggttgtccaccuuu a 21 9 21 DNA Artificial Sequence antisense oligonucleotide 9ucagugcttt gatggcguag u 21 10 21 DNA Artificial Sequence antisenseoligonucleotide 10 cuucucgcag tacaggcucu c 21 11 21 DNA ArtificialSequence antisense oligonucleotide 11 caagacgctt gtgcacguuu a 21 12 21DNA Artificial Sequence antisense oligonucleotide 12 ucagagcttagttggcguug u 21

What is claimed is:
 1. A method of treating a disease or pathologicalcondition associated with dysregulation of the PI-3 kinase pathway,comprising administering to a subject suffering from said disease aneffective amount of a composition that inhibits the activity of proteinkinase N beta.
 2. The method according to claim 1, wherein saiddysregulation of said PI-3 pathway is associated with increased activityof protein kinase N beta.
 3. The method according to claim 1, whereinsaid disease or pathological condition is cancer or a precancerousgrowth.
 4. The method according to claim 3, wherein said disease orpathological condition is selected from the group consisting ofendometrial cancer, colorectal carcinoma, glioma, endometrial cancer,adenocarcinoma, endometrial hyperplasia, Cowden's syndrome, hereditarynon-polyposis colorectal carcinoma, Li-Fraumeni syndrome, breast cancer,thyroid cancer, ovarian cancer, and prostate cancer.
 5. The methodaccording to claim 2, wherein said disease of pathological condition isselected from the group consisting of Bannayan-Zonana syndrome,Lhermitte-Duklos′ syndrome, a hamartoma-macrocephaly diseases, amucocutaneous lesion, macrocephaly, mental retardation, gastrointestinalhamiatoma, lipoma, thyroid adenomas, fibrocystic disease of the breast,and cerebellar dysplastic gangliocytoma.
 6. The method according toclaim 1, wherein said composition comprises at least one agent selectedfrom the group consisting of a peptide, a protein, an antibody, ananticaline, a functional nucleic acid, and a small molecule drug.
 7. Themethod according to claim 6, wherein said agent is a functional nucleicacid selected from the group consisting of an aptamer, an aptazyme, aribozyme, a spiegelmer, an antisense oligonucleotide and an siRNA. 8.The method according to claim 7, wherein said agent is an antisenseoligonucleotide having a sequence selected from the group consisting ofSEQ ID NOS 1-12.
 9. The method according to claim 7, wherein said agentis an siRNA comprising a sequence selected from the group consisting of5′ actgagcaagaggctttggag and 5′ aaattccagtggttcattcca.
 10. The methodaccording to claim 1, wherein said subject is a human.
 11. A method foridentifying an agent suitable for treating a disease or pathologicalcondition associated with dysregulation of the PI-3 kinase pathway,comprising contacting a test system comprising a protein having proteinkinase N beta activity with a composition comprising a candidatecompound, and determining if protein kinase N beta activity is reducedin the presence of said candidate compound.
 12. The method according toclaim 11, wherein reduction in protein kinase N beta activity ismeasured by measuring protein kinase N beta enzymatic activity.
 13. Themethod according to claim 11, wherein reduction in protein kinase N betaactivity is measured by measuring a change in expression of said proteinhaving protein kinase N beta activity.
 14. The method according to claim11, wherein said test system comprises a cell that expresses saidprotein having protein kinase N activity.
 15. The method according toclaim 11 wherein said candidate compound is selected from the groupconsisting of a peptide, a protein, an antibody, an anticaline, afunctional nucleic acid, a small molecule drug, an aptamer, an aptazyme,a ribozyme, a spiegehner, an antisense oligonucleotide and an siRNA. 16.The method according to claim 11, wherein said composition comprises aplurality of candidate compounds.
 17. A method for diagnosing a diseaseassociated with a dysregulated PI-3 kinase pathway in a subjectsuspected of suffering from said disease, comprising measuring proteinkinase N beta activity in a sample obtained from said subject andcomparing said activity with a control level of activity, wherein anincrease in protein kinase N beta activity indicates the presence ofdisease.
 18. The method according to claim 17, wherein protein kinase Nbeta activity is measured by determining expression of protein kinase Nbeta protein.
 19. The method according to claim 17, wherein proteinkinase N beta activity is measured by determining enzymatic activity ofprotein kinase N beta.
 20. The method according to claim 17, whereinsaid control level of activity is measured in a control tissue obtainedfrom said subject and wherein said control tissue is not suspected ofhaving a dysregulated PI-3 kinase pathway.
 21. The method according toclaim 17, wherein said control level of activity is taken from adatabase of control levels.
 22. A method for determining the efficacy ofa therapeutic treatment regimen in a subject, comprising: measuringprotein kinase N beta activity in a first sample obtained from thesubject, thereby generating an initial level; administering thetreatment regimen to the subject; measuring protein kinase N betaactivity in a second sample from the patient at a time followingadministration of the treatment regimen, thereby generating a testlevel; and comparing the initial and test levels, wherein a decrease inprotein kinase N beta activity in the test level relative to the initiallevel indicates that the treatment regimen is effective in the patient.23. A method for selecting test agents having a therapeutic effect in asubject, comprising: measuring protein kinase N beta activity in a firstsample obtained from the subject, thereby generating a pre-treatmentlevel; administering a test agent to the subject; measuring proteinkinase N beta activity in a second sample from the patient at a timefollowing administration of the test agent, thereby generating data fora test level; and comparing the pre-treatment level to the test level,wherein data showing no decrease in the test level relative to thepre-treatment level indicates that the test agent is not effective inthe patient; and eliminating the test agent from further evaluation orstudy.
 24. A pharmaceutical composition comprising at least one agentthat inhibits the activity of protein kinase N beta and apharmaceutically acceptable carrier.
 25. The composition according toclaim 24, wherein said agent is selected from the group consisting ofagents that inhibit the enzymatic activity of protein kinase N beta. 26.The composition according to claim 24, wherein said agent is selectedfrom the group consisting of agents that inhibit the expression ofprotein kinase N beta.
 27. The composition according to claim 24,wherein said agent is selected from the group consisting of smallmolecules that interact with protein kinase N beta, antibodies thatspecifically bind protein kinase N beta, polypeptides that bind toprotein kinase N beta, and functional nucleic acids.
 28. The compositionaccording to claim 27, wherein said functional nucleic acid is selectedfrom the group consisting of an aptamer, an aptazyme, a ribozyme, aspiegelmer, an antisense oligonucleotide and an siRNA.